KR101530498B1 - A method for detecting the methylation rate of target gene as screening marker - Google Patents

Abstract

A method for cancer detection using the methylation rate as an examination index for detecting a methylation rate of a target gene of a specimen and determining a risk of having cancer, the method comprising the steps of: Provide; In step 2, the methylation rate of the CpG sequence is detected in at least one target gene of the specimen, wherein the target gene is selected from the group consisting of PTPRR (protein tyrosine phosphatase, receptor type R), ZNF582 (zinc finger protein 582), phosphodiesterase 8B ) And DBC1 (deleted in bladder cancer protein 1); In step 3, it is determined whether there is or is not a pathological change of cancer or precancer in the specimen depending on the methylation rate in the target gene, or the methylation rate of the target gene is used as a prognostic marker.

Description

A method of detecting the methylation rate of a target gene as a test marker {A METHOD FOR DETECTING THE METHYLATION RATE OF TARGET GENE AS SCREENING MARKER}

The present invention relates to a method for screening cancer, a biomarker for screening cancer, and uses thereof. In particular, the present invention relates to a method for testing cancer using methylated DNA as a biomarker.

Cervical cancer has become one of the leading causes of death among women worldwide and in Taiwan. According to a 2002 statistical survey by the International Health Organization, cervical cancer was the second leading disease responsible for the deaths of women worldwide after breast cancer. Regular cervical cancer screening is the best way to prevent cervical cancer. Conventional cervical cancer screening includes two approaches: the most commonly used Pap smear and human papilloma virus testing (HPV testing). The Pap test is to detect cervical cancer early by sampling secretions from the cervix uteri and examining whether there is a cancerous pathological change in the exfoliated epithelial cells under the microscope. It consists of doing. On the other hand, the human papilloma virus test uses a polymerase chain reaction (PCR) or hybrid capture to test whether human papilloma virus (HPV) is present in the target. Consists of

However, there are a number of undesirable properties for cytology. For one, it requires collection by a physician and analysis by a medical examiner/pathologist, which in many developing countries poses a high cost of manpower that imposes difficulties in facilitating this examination. Demand. In addition, the Pap test has a high false negative rate, which delays diagnosis and appropriate treatment prior to pathological changes in cancer. For human papilloma virus testing, although it is highly sensitive, it tends to produce a high false positive rate, which not only leaves patients vain anxiety, but also in the test follow-ups of these false-positive patients. So it wastes a lot of medical resources. Therefore, one of the most important topics in facilitating cervical cancer screening is to increase the accuracy and convenience of cervical cancer screening methods.

Genomic deletions have long been considered an important factor in tumorigenesis. For a long time, I have been accustomed to the idea that the coding potential of the genome lies in the sequence of four bases A, T, G, and C. The two-hit theory proposed in the early 1970s indicates that accompanying mutations or deletions of some homologous tumor suppressor genes cause or predispose to the development of cancer. However, separate information affecting the phenotype may be stored in the modified base 5-methylcytosine. 5-methylcytosine is found in mammals within the context of the palindromic sequence 5'-CpG-3'. Most CpG dinucleotide pairs are methylated in mammalian cells except for some regions called "CpG islands". CpG islands are usually present in approximately 1 kb of GC- and CpG-rich regions located in the vicinity of genes, and are also found near the promoter of widely expressed genes. Cytosine methylation is methyl donor (methyl donor) S after DNA synthesis-adenosyl-methionine takes place by the enzymatic transfer of the erosion carbon -5 position (carbon-5 position) of the methyl groups from cytosine (S -adenosylmethionine). The enzymatic reaction is carried out by DNA methyltransferases (DNMTs). DNMT1 is a major methyl group transferase in mammals and is responsible for post-replicative restoration of hemi-methylated sites referred to as maintenance methylation. On the other hand, DNMT3A and DNMT3B are believed to be primarily involved in the methylation of new sites, a process called isolated methylation.

The loss of methylation in CpG dinucleotides, ie normal hypomethylation, is the first epigenetic abnormalities identified in cancer cells. However, over the past few years, it has become increasingly apparent that site-specific hypermethylation, such as some tumor suppressor genes, can provide selective benefits during carcinogenesis. have. Dense methylation of CpG islands in the promoter regions can trigger chromatin remodeling through histone modifications followed by gene silencing. Thus, in addition to chromosomal deletions or genetic mutations, epigenetic silencing of tumor suppressor genes by promoter hypermethylation is commonly seen in human cancers.

A recent epidemiologic study revealed a correlation between serum folate levels, a major source of methyl groups, and human papilloma virus infection and clearance. Genetic polymorphisms of enzymes in the metabolism of the methyl cycle have also been reported to be associated with the development of cervical intraepithelial lesions. As the concept of epigenetics has developed, studies exploring the link between DNA methylation and cervical cancer have also made rapid progress. Studies of DNA methylation in cervical cancer are accumulating, indicating the possibility of using methylation as an indicator in the examination of the uterus. As for the nature of the interface between genetics and the environment, the prevalence of methylation in tumor suppressor genes varies in different genes and with different distributions. The concept of methylator phenotypes with different disease behaviors has been proposed as a matter of debate. The methylator phenotype of cervical cancer and its interaction with human papilloma virus genotypes remain unknown. Which genes are specifically methylated in cervical cancer and how many genes are required to achieve clinical application will remain a challenge for the coming future.

The inventors of the present application are Taiwan (Taiwan Patent Publication No. 200831900), China (Chinese Patent Application No. 200810094659.2), Malaysia (No. UI20085354) and the United States (United States Patent Publication No. 20080311570) (hereinafter referred to as'prior applications). ') has applied for related patent applications. Compared with previous applications, the present inventors have discovered some novel cancer screening biomarkers and cancer screening methods using the same.

The present invention aims to provide a method for screening cancer, a biomarker for screening cancer, and a use thereof, and in particular, cancer using methylated DNA as a biomarker It aims to provide a method for testing.

One object of the present invention is to provide a method for testing cervical cancer as a first line cervical cancer screening.

Another object of the present invention is characterized in that it can be used not only in the first cervical cancer test, but also in the second cervical cancer test to assist in the human papilloma virus test or the uncertain cytology test result in order to achieve a more accurate cervical cancer test effect. It is to provide a method for cervical cancer screening.

Another object of the present invention is that it can be used in the examination of other cancers (e.g.: ovarian cancer, colon cancer, etc.) to aid in the diagnosis of an abnormal specimen, as in the examination of cervical cancer. It is to provide a cancer screening method.

Another object of the present invention is to provide a cancer test biomarker or a kit comprising the biomarker for testing the risk of having cancer.

In order to achieve the above-mentioned objects of the present invention, in the cancer screening method using the methylation rate as a test index for detecting the methylation rate of the target gene of the specimen and determining the risk of having cancer, the method comprises the following And, in step 1, providing a specimen; In step 2, the methylation rate of the CpG sequence is detected in at least one target gene of the specimen, and the target gene is PTPRR (protein tyrosine phosphatase, receptor type, R), ZNF582 (Zinc Finger Protein 582), PDE8B (phosphodiesterase 8B). ) And DBC1 (deleted in bladder cancer protein 1); In step 3, depending on the rate of methylation in the target gene, it is determined whether or not there is a pathological change of cancer or precancer in the specimen, or the rate of methylation of the target gene is used as a prognostic marker.

In a preferred embodiment, the specimen is isolated selected from the group consisting of cervical smear, ascites, blood, urine, excrement, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells, and postoperative cancer tissue. It may be a psalm that has been made.

In a preferred embodiment, the test method for the methylation state of the CpG sequence of the target gene is methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (QMSP). ; quantitative methylation-specific polymerase chain reaction), bisulfite sequencing (BS), microarray, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing Includes.

In a preferred embodiment, the target gene DBC1 (deleted in bladder cancer protein 1) has a nucleotide sequence of SEQ ID NO: 1, the target gene PDE8B (phosphodiesterase 8B) has a nucleotide sequence of SEQ ID NO: 2, and the target gene PTPRR ( Protein tyrosine phosphatase, receptor type, R) has a nucleotide sequence of SEQ ID NO: 3, and the target gene ZNF582 (Zinc Finger Protein 582) has a nucleotide sequence of SEQ ID NO: 4.

In a preferred embodiment, the methylated bisulfite sequencing of the target gene DBC1 (deleted in bladder cancer protein 1) is recognized by the primers of SEQ ID NOs: 13 and 14, and the target gene PDE8B (phosphodiesterase 8B) is SEQ ID NO: 15, Recognized by the primers of 16, the target gene PTPRR (protein tyrosine phosphatase, receptor type, R) has a nucleotide sequence of SEQ ID NOs: 17 and 18, and the target gene ZNF582 (Zinc Finger Protein 582) is SEQ ID NO: 19, 20 Has a nucleotide sequence of.

In a preferred embodiment, the target gene DBC1 (deleted in bladder cancer protein 1) is recognized by the primers of SEQ ID NOs: 5 and 6, and the target gene PDE8B (phosphodiesterase 8B) is recognized by the primers of SEQ ID NOs: 7 and 8. , The target gene PTPRR (protein tyrosine phosphatase, receptor type, R) has a nucleotide sequence of SEQ ID NOs: 9 and 10, and the target gene ZNF582 (Zinc Finger Protein 582) has a nucleotide sequence of SEQ ID NOs: 11 and 12.

The cancer may be selected from the group consisting of cervical cancer, colon cancer, and ovarian cancer.

The term "test specimen" refers to an isolated test specimen in the present specification, and the isolated test specimen includes cervical debris, ascites, blood, urine, excrement, sputum, oral mucosa cells, gastric juice, bile , Cervical epithelial cells, postoperative cancer tissue, or other suitable specimens. The cancer testing method of the present invention is useful for testing the methylation status of target genes in the specimens, and uses the methylation status of a biomarker as a test index for various types of cancer. The cancer screening method and the test index of biomarkers provided by the present invention can be used by researchers to perform the test in a laboratory.

As used herein, "the risk of having cancer" refers to the percentage of suffering from cancer, the risk of developing cancer in the future, or the presence or absence of cancer.

If the methylation status is higher compared to the appropriate control of the target gene (biomarker), the risk of having cancer may be higher.

According to the above test method, the present invention also includes a cancer test biomarker or a kit for testing the methylation status of a target gene (biomarker) in an isolated specimen to determine the risk of having cancer. to provide.

These features and advantages of the present invention will be fully understood and recognized from the detailed description of the accompanying drawings below.

The present invention provides an effect of providing a method for screening cancer, a biomarker for screening cancer, and uses thereof, and in particular, screening for cancer using methylated DNA as a biomarker It provides the effect of providing a way to do it.

1A is a diagram showing the results obtained by performing bisulfite sequencing (BS) on several cervical specimens using the target gene PTPRR in the cancer testing method of the present invention.
1B is a diagram showing the results obtained by performing bisulfite sequencing (BS) on several cervical specimens using the target gene ZNF582 in the cancer testing method of the present invention.
1C is a diagram showing the results obtained by performing bisulfite sequencing (BS) on several cervical specimens using the target gene PDE8B in the cancer testing method of the present invention.
1D is a view showing the results obtained by performing bisulfite sequencing (BS) on several cervical specimens using the target gene DBC1 in the cancer testing method of the present invention.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Materials and methods

1. Materials

Test materials included a series of complete cervical lesion specimens, including squamous cell carcinoma (SCC, n = 20), adenocarcinoma (AC, n = 20) and normal cervical specimens (n = 10). Included. All of these cervical specimens, ovarian specimens and colon cancer specimens were obtained from Tri-service General Hospital, Taipei, Taiwan. Each specimen was subjected to genomic DNA extraction by means of the QIAamp DNA Kit (QIAGEN), and in genome-wide DNA methylation. Used in the control and analysis of DNA methylation conditions. Prior to the analysis, the quality of genomic DNA was checked by a bioanalyzer (Agilent). In this Example, 10 µg of fragmented DNA was subjected to Medip (MeDIP; methyl DNA IP) analysis.

2. Medif and CpG Island-Plus-Promoter Array ( CpG island - Plus - Promoter DNA methylation analysis by arrays)

Genomic DNA was fragmented into a size ranging from 300 to 1,000 bp with Bioruptor™ UCS-200 (Diagenode). The present inventors used 100 μl IP buffer (0.15% SDS (sodium dodecyl sulfate), 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris, and 0.1% BSA. In the final concentration of (bovine serum albumin)), it was immunoprecipitated overnight at 4° C. with 30 μl of a polyclonal anti-5′-methyl cytosine antibody (Abcam). The present inventors incubated a mixture with 120 µl of Protein G Sepharose (Amersham) at 4° C. for 2 hours, and then incubated with 1 ml of a low concentration of salt, a high concentration of salt buffer, and lithium chloride. And twice with TE buffer. Subsequently, the present inventors treated the protein paper twice with an elution buffer (elution buffer; 1% SDS, 0.1M NaHCO ₃ ) at room temperature for 15 minutes, and methylated by phenol-chloroform extraction and subsequent ethanol precipitation. The resulting DNA was recovered. The enriched, methylated DNA and input DNA were amplified with a Whole Genome Amplification Kit (Sigma). The enriched total DNA was end-labeled with Cy5 and Cy3, respectively, and CpG island-plus-promoter arrays designed and synthesized by NimbleGen Systems, Inc. It was co-hybridized. This array contained the double present 24,659 HG18 RefSeq promoters (800 bp upstream to 200 bp downstream) and 385,000 50-75 bp oligonucleotides tiled every 100 bp across 28,226 CpG islands.

Each feature on the array has a corresponding scaled log2-ration. The log2-ratio is a normalization to the center of the ratio data in the vicinity of zero. From the scaled log2-ratio data, a fixed-length window (500bp) is positioned around each successive probe and is one-sided Kolmogorov-Smirnov (KS). A test was applied to determine whether the probes were taken from a significantly more positive distribution than the probes in the rest of the array. The resulting score for each probe is the -log10 p-value from the KS test with a window around the probe (see 2006 by Scacheri and his colleagues). The present inventors detected peaks enriched by searching for at least two probes that were equal to or greater than a p-value minimum cutoff of 2 (-log10). Different peaks within 300bp were merged. Differential methylation regions between SCC/AC and normal cervix within 2500bp upstream of transcription star sites were selected for future identification. Finally, the p-value data was observed using a signal map (SignalMap; NimbleGen).

3. Bisulfite denaturation, methyl -specific polymerase chain reaction ( MSP ) and bisulfite sequencing (BS)

DNA modification kit (Ternecula, CA, USA) according to the manufacturer's recommendations to convert 1 μg aliquots of genomic DNA to sodium bisulfite to preserve methylated cytosines. , CA) material of Chemicalcon (Chemicon)) was used. The final precipitate was eluted with 70 μl of pre-warmed TE buffer for MSP.

Normal DNA from human peripheral blood was taken and subjected to bisulfite denaturation, which was used as a control with an unmethylated promoter sequence.

MSP was performed according to the prior art. In summary, 1 μl of denatured DNA was amplified using MSP primers (Table 1) that specifically recognize methylated gene sequences present in bisulfite-converted DNAP. 1 μl of denatured template DNA, 1.5 pmol of each primer, 0.2 mmol/L deoxynucleotide triphosphates and 1 unit of Gold Taq DNA polymerase (Foster, CA, USA) Methylation-specific polymerase chain reaction was carried out in a total volume of 25 µl containing Applied Biosystems Co., Ltd. MSP reactions were subjected to initial incubation at 95°C for 5 minutes, followed by 35 cycles were performed at 95° C. for 30 seconds, and annealing was performed for 30 seconds at 72° C. at an appropriate temperature for 30 seconds, and final extension was performed at 72° C. for 5 minutes. bromide) was visualized on a 2.5% agarose gel, and illuminated under UV light.

Table 1 is a sequence of MSP primers.

All MSP data were obtained from at least two independent modifications of DNA. The absence of a signal in duplicate experiments was recorded as negative methylation. A PCR product obtained by using an MSP primer (M) capable of specifically recognizing a methylated gene sequence was constructed into a pCR4-TOPO vector (Invitrogen, Carlsbad, CA). Five independent clones were selected and subjected to bisulfite sequencing (BS). Primers used in bisulfite sequencing (BS) are listed in Table 2. It was performed using a 377 Auto-sequencer (Applied Biosystems, Foster, CA, USA). The sequenced results are shown in the sequence list. In bisulfite sequencing, the sequence numbers were DBC1_BS (SEQ ID NO: 21), PDE8B_BS (SEQ ID NO: 22), PTPRR_BS (SEQ ID NO: 23) and ZNF582_BS (SEQ ID NO: 24), respectively.

Table 2 shows the sequences of bisulfite sequencing primers.

Example 2 Test for methylated target genes in cervical cancer

After testing by CpG island-plus-promoter arrays, four target genes that would have been highly methylated in cervical cancer cells, namely DBC1 (SEQ ID NO: 1), PDE8B (SEQ ID NO: 2), PTPRR (SEQ ID NO: 2), respectively. : 3) and ZNF582 (SEQ ID NO: 4) were examined. Their detailed information is shown in Table 3. From Table 3, among these four genes, few studies have shown the correlation of these genes for cervical cancer except for DBC1, which is known to be associated with bladder cancer.

Table 3 shows the properties of methylated genes in cervical cancer identified using CpG island-plus-promoter microarray.

Example 3 Analysis of methylation status of target genes in cervical lesion specimens by bisulfite sequencing ( BS)

Target gene: PTPRR

Psalms groups:

1. HeLa_0: HeLa cervical cancer cell line;

2. HeLa_10D: HeLa cervical cancer cell line (HeLa_0) treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC);

3. HeLa_DT: 10 μM DNA methyltransferase inhibitor 5′-aza-2′-deoxycytidine (AZC) and 0.33 μM TSA (Sigma Chemical Co., St. Louis, Mo.) Treated HeLa cervical cancer cell line (HeLa_0);

4. AC: cervical adenocarcinoma specimen;

5. SCC: cervical squamous cell carcinoma specimen;

6. Control (normal): Normal cervical blood DNA as a control without methylation.

Each of these specimens described above was subjected to bisulfite sequencing (BS) following bisulfite denaturation to analyze whether the target gene (PTPRR) in each specimen was hypermethylated or not. The results are shown in FIG. 1, in which black indicates a methylation region and white indicates an un-methylation region. The target gene PTPRR was not methylated in the control and the adenocarcinoma, whereas the target gene PTPRR was hypermethylated in the HeLa cervical cancer cell line (HeLa_0) and the cervical squamous cell carcinoma specimen (SCC). Therefore, the methylation rate of PTPRR can be used as a biomarker for testing whether cervical cancer is present or not.

In addition, in order to confirm whether the methylation rate of the target gene in the cervical cancer specimen can be regulated through DNA methylation, the HeLa cervical cancer cell line was used as a 10 mM DNA methyltransferase inhibitor 5'-aza-2'-deoxy. It was treated with cytidine (AZC; Sigma Chemical Company). The DNA specimen extracted from the cells was subjected to bisulfite sequencing (BS) following bisulfite denaturation. The results shown in FIG. 1A show that the target gene PTPRR in the AZC-treated HeLa cervical cancer cell line (HeLa_10D) was de-methylated compared to the cervical squamous cell carcinoma specimen (SCC) and the HeLa cervical cancer cell line (HeLa_0). It has been shown to have a portion of the de-methylated) region.

Tricostatin A (TSA) is a histone deacetylase (HDAC) inhibitor and can be used to lower or degrade the methylation rate. The HeLa cervical cancer cell line (HeLa_DT) was treated with both AZC and TSA. The results shown in FIG. 1 show that the target gene PTPRR in the AZC- and TSA-treated HeLa cervical cancer cell line (HeLa_DT) was higher than that of the cervical squamous cell carcinoma specimen (SCC) and the HeLa cervical cancer cell line (HeLa_0). It was shown to be de-methylated.

In summary, the above-described results confirm that the target gene PTPRR in cervical cancer specimens was methylated through DNA methylation.

Target gene: ZNF582

Psalm groups:

1. HeLa_0: HeLa cervical cancer cell line;

2. HeLa_10D: HeLa cervical cancer cell line (HeLa_0) treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC);

3. HeLa_DT: HeLa cervical cancer cell line (HeLa_0) treated with both 10 μM DNA methyltransferase inhibitor 5′-aza-2′-dioxycytidine (AZC) and 0.33 μM TSA;

4. SCC1: cervical squamous cell carcinoma specimen 1;

5. SCC2: cervical squamous cell carcinoma specimen 2;

6. Control (normal): Normal cervical blood DNA as a non-methylated control.

The cervical squamous cell carcinoma specimen 1 and specimen 2 are specimens obtained from different patients.

These specimens mentioned above were subjected to bisulfite sequencing (BS) to analyze whether the target gene (ZNF582) in each specimen was hypermethylated or not. The results shown in FIG. 1B show that the target gene ZNF582 in the cervical squamous cell carcinoma specimen 1 (SCC) and cervical squamous cell carcinoma specimen 2 (SCC) and the HeLa cervical cancer cell line (HeLa_0) was hypermethylated compared to the control group. Indicated. Thus, the target gene ZNF582 in cervical cancer specimens was highly methylated.

Target gene: PDE8B

Psalm groups:

1. SiHa_0: SiHa cervical cancer cell line;

2. SiHa_10D: SiHa cervical cancer cell line (SiHa_0) treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-dioxycytidine (AZC);

3. SiHa_DT: SiHa cervical cancer cell line (SiHa_0) treated with both 10 μM DNA methyltransferase inhibitor 5′-aza-2′-dioxycytidine (AZC) and 0.33 μM TSA;

4. SCC1: cervical squamous cell carcinoma specimen 1;

5. SCC2: cervical squamous cell carcinoma specimen 2;

6. Control (normal): normal cervical blood DNA used as a non-methylated control;

The cervical squamous cell carcinoma specimen 1 and specimen 2 are specimens obtained from different patients.

These specimens mentioned above were subjected to bisulfite sequencing (BS) to analyze whether the target gene (PDE8B) in each specimen was hypermethylated or not. The results shown in FIG. 1c show that the target gene PDE8B in cervical squamous cell carcinoma specimen 1 (SCC) and cervical squamous cell carcinoma specimen 2 (SCC) and SiHa cervical cancer cell line (SiHa_0) was hypermethylated compared to the control group. Indicated. Thus, the target gene PDE8B in cervical cancer specimens was highly methylated.

Target gene DBC1

Psalm groups:

1. SiHa_0: SiHa cervical cancer cell line;

2. SiHa_10D: SiHa cervical cancer cell line (SiHa_0) treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-dioxycytidine (AZC);

3. SiHa_DT: SiHa cervical cancer cell line (SiHa_0) treated with both 10 μM DNA methyltransferase inhibitor 5′-aza-2′-dioxycytidine (AZC) and 0.33 μM TSA;

4. SCC1: cervical squamous cell carcinoma specimen 1;

5. SCC2: cervical squamous cell carcinoma specimen 2;

6. Control (normal): normal cervical blood DNA as a non-methylated control;

The cervical squamous cell carcinoma specimen 1 and specimen 2 are specimens obtained from different patients.

These specimens mentioned above were subjected to bisulfite sequencing (BS) to analyze whether the target gene (DBC1) in each specimen was hypermethylated or not. The results shown in FIG. 1D show that the target gene DBC1 in the cervical squamous cell carcinoma specimen 1 (SCC) and cervical squamous cell carcinoma specimen 2 (SCC) and the SiHa cervical cancer cell line (SiHa_0) was hypermethylated compared to the control group. Indicated. Therefore, the target gene DBC1 in cervical cancer specimens was highly methylated.

Example 4 Methylation analysis of the target gene in cervical cancer specimens

Methylation-specific PCR (MSP) was used to analyze the methylation status of the four target genes in cervical squamous cell carcinoma (SCC) specimens. The methylation status analysis results shown in Table 4 show that the methylation frequencies of DBC1, PDE8B, PTPRR, and ZNF582 were 11%, 0%, 9% and 6%, respectively, in normal cervical specimens; In cervical squamous cell carcinoma specimens, the methylation frequencies of DBC1, PDE8B, PTPRR and ZNF582 were 100%, 47%, 100% and 97%, respectively. From these results, it can be seen that the four genes were highly methylated in the cervical squamous cell carcinoma specimens. Therefore, in the end, the methylation rates of DBC1, PDE8B, PTPRR and ZNF582 can be used as test indexes in the examination of cervical cancer.

Table 4 is an analysis of the methylation status of the target gene in cervical squamous cell carcinoma specimens.

Example 5 Methylation analysis of the target gene in ovarian cancer specimens

Methylation-specific PCR (MSP) was used to analyze the methylation status of the target gene in ovarian cancer specimens. The methylation status analysis results in Table 5 report the analysis of the methylation status of the three genes DBC1, PTPRR, and ZNF582 in the ovarian malignant tumor specimen and the ovarian benign tumor specimen. The results indicated that the methylation frequencies of DBC1, PTPRR, and ZNF582 in ovarian malignant tumor specimens were 50.3%, 50.0% and 56.3%, respectively, and the methylation frequencies of DBC1, PTPRR and ZNF582 in ovarian benign tumor specimens were 2.5%, 0.0%, and respectively. 12.5%. The differential methylation levels were 53.8%, 50.0% and 43.8%, respectively. Thus, compared to the ovarian benign tumor specimen, the methylation levels of the three genes in the ovarian malignant tumor specimen were significantly higher. Therefore, the methylation rate of DBC1, PTPRR and ZNF582 can be used as a test index in ovarian cancer screening.

Table 5 is an analysis of the methylation rate of the target gene in ovarian cancer specimens.

Example 6 Methylation analysis of the target gene in colon cancer specimens

Methylation-specific PCR (MSP) was used to analyze the methylation status of the target gene in colon cancer specimens. The methylation status analysis results in Table 6 report the methylation status of the four genes DBC1, PDE8B, PTPRR, and ZNF582 in the colon cancer specimen. The results show that the methylation frequencies of DBC1, PDE8B, PTPRR and ZNF582 in colon cancer specimens were 100.0%, 100.0, 100.0% and 100.0%, respectively, whereas the methylation frequencies of DBC1, PDE8B, PTPRR and ZNF582 in normal colon tissue specimens were 25.0, respectively. %, 25.0%, 25.0% and 25.0%. Thus, compared to the normal colon tissue specimen, the methylation levels of the four genes in the colon cancer specimen were markedly higher. Therefore, the methylation rate of DBC1, PDE8B, PTPRR and ZNF582 can be used as a test index in colon cancer screening.

Table 6 is an analysis of the methylation status of the target gene in colon cancer specimens.

The cancer screening method provided by the present invention has the following advantages over the conventional techniques described above:

1. The cancer testing method, biomarker and use thereof provided by the present invention use the methylation rate of a specific gene in the isolated specimen as a biomarker for determining the risk of having cancer, and use cervical scraping. ) And human papilloma virus test (HPV test), and the like, both the sensitivity and specificity of the cancer screening method of the present invention are higher than those of the two described methods.

2. The cancer screening method, biomarker and use thereof provided by the present invention are not only in the primary cervical cancer test, but also as a secondary cervical cancer test, together with or assisting with the human papilloma virus test, thereby providing a more accurate cervical cancer test effect. Can be achieved.

3. The cancer test method, biomarker and use thereof provided by the present invention are applied not only to the test for cervical cancer, but also to test for other cancers (for example: ovarian cancer, colon cancer, etc.) to aid in the diagnosis of abnormal specimens. can do.

Of course many changes and changes in the previously described embodiments of the present invention can be implemented without departing from its point of view. Accordingly, in order to facilitate the development of science and useful arts, the present invention has been described and is intended to be limited only by the appended claims.

<110> National Defense Medical Center <120> Cancer Screening Method <130> <140> <141> <160> 24 <210> 1 <211> 5000 <212> DNA <213> Homo sapiens <400> 1 ggtcttcacc tgaagttgcc tgactacgtt gtctcagatt gatttcagaa ctgcattatc 60 agcagggcag ctgttgccta ctaacagtca tggaagtaga tctcagctgg gggatgatgg 120 ggaaacaaag agaaagatag ggacgaataa aggaaggaat ggggcacaga cagaaagaga 180 gagagagaaa agaaagaaag gaggagaggc aagagatagt gacctggggt ttcataccgc 240 ggtgggtggg agaaggagtg tgtgtggtgg gcgtatttct tcgtggtcag aatcaaagtc 300 agaactgacc tggggaacct gtgttcaaga cctgaacctg gaccagaagc caagggaaag 360 agaaagttgc ccaagaaaaa agggagaaat ttccaccagg cgaaaataga aatcgctgac 420 tgggtttgtg gctggagagc tgtccgtggt gctgactcct cctcattggg attccagtga 480 aggctgagaa gctctgcact ggctgctcct cctctccctt tcctctcccc tcagtcctga 540 ccttctgaga cccaggatca ttctggctag ctcgatgtct tctcctctct ttctcccttt 600 ctctttgtcg attaaataat ttttcccgag ttcccagttc atcactcaaa catttctctt 660 ccattttgtt tcaagactcc tcttcctcca attagttcac tggctcctag attgttctag 720 gcctccctgg tggtcacatc tctctcttat catcctttga attaaaaaac aaacaaaaaa 780 catacaatgg caatcataaa agcagacaac atgcactgag cactcactag gtgtcaggga 840 gcagtctaag tgcctttgca tacattaact catgagcagt ctaagtgcct ttgcatatat 900 taactcatta caattccaca tcaaccctat tagatgggta gatatattat tattctatta 960 aacagacaag gaattgaggc ttaaggggta agtgatttca ccaagaccag acagctacag 1020 gactccaacc tctctggagc ctgcacttct actgaatctg gctcttttac atattccaac 1080 accttttcct atgacttcct gcccctccat ctggtagcct taatgctttc tattcttgcc 1140 taatcttcag gggactaggg atctgcattg ctgatttggt ttaaatcaga agggaagcaa 1200 agtaaacaaa cataccaaat gtgcgctgtg tgggaattat catacgaggg ctttattttc 1260 tgcttcagga agaggcccta tgttagcagc cccagcctgc attcaggctg attgcagagt 1320 attttgcttt ttattttcat gtcttagtcc ctgtaccctc gccccttccc cgcctctggt 1380 ggtctccaga gaacttcgtg tcccctcagc ttctccctcc tacatcctgc ctacgtagag 1440 aagctcttgc ttcattctgg gaggttacgt gggctctcgc ctacacaccg agagaaacaa 1500 acagtgtcaa acactcacag agagacgcgc agacacaaac ggacccacac gggcaactcc 1560 cgagacaaaa cccacactcg atggatccac gcggccgtgg aaacacctgc cgccccagaa 1620 acactcaggt actcgcgaca cacacagtac agtcacgctt aagggcacca ggattccggg 1680 tttgcgcgta tgcgcggtcc ctttggatgc tcgtgcgcat agacacaaca ccctacacgc 1740 cccagaccca cgaaactccc tacggctcag ccccagccca cccgggccgc ccttccctcg 1800 aggcggcctc ccgtctctcc tcctctcgct tctcctcctc ctccgcctaa agatgtacaa 1860 aacactcctc ggaagcaacc ccggcgttca gctcctccct ccccgccccc cggccgccgc 1920 tcccccattc attttcggcc gtcgccggct aagtccctcc cccggcgtag cccggcctcc 1980 gccgctcccc gcccggagac cgcggcgcac ttggacttcc ctctccattc gccagccgcc 2040 tcgctcccgg accccacggc tgcaaactga tctggcgcgc ggggaggagg agagcgcagg 2100 cgagcgaacc cgcgagagag ggagagagcg agcgagcaac agcgagagcg agagcgagag 2160 agccgggagg cagagggagt agtgaccgcc ttccggagcc gggattcatg cctgtcctcg 2220 ggaccagcga aggggacttt acggctgagt atgagccagg ctgctaggag ccaggtaccc 2280 ccacgcctgc agtccccgcg ccgtgcccgg aatgcgagct gcacgcaggg ctctcccaag 2340 ttcccaccga gccgaataaa aagcgtcctc ccgcagctct ccgccaaaga cggacattga 2400 ctccaggtaa ggcggcgccg ggtgcagcgc cccgcagccc cgctgccctt ggacccggcc 2460 ccgggccgca ttcggggcgt ccccgcgctc ctctgcccct ccccctaccg gcacccttgt 2520 ccgctcttca cctggccgcc ccgccgcctc caagtcttct ccagttctag ggagggggtt 2580 cctgtgcctg gggctcaaag ggctaattgc gggtttgagt gagtggcgtg tgtgtccccg 2640 cgcgctcccg acgtgtgcac catggtggga acttgatgtg gtgctagtgt gtttgcgtgt 2700 gcggcgtcgt ttgcgtttga tgcgtgtgtt cgtggtgtgt gtgtgtgtcc gtgtgtgtaa 2760 gggaggggtg aagagagaga ggtcctataa cctacttacg gcgcgatgtg tgtgtgcatg 2820 tttgtacgta tgtgtttgta tgtgtgccct cgtgtgtctt tttaattagg tctctccagc 2880 ttacacggaa tgggaccctt actataggat cacgtagtca ccgggaaacc cgctgtggac 2940 ttcctcttgg ggctctgggc ttggggtttg gggaggatta tggggctgta gatggcacct 3000 tatttagccc aatgttggta cgcttgaagg aaaattcctc caaacggtgg aatcctgcta 3060 cactgggacc cacagcttaa tatgaaagag acatggccaa cccccgaggc aaatgagacg 3120 ctgtcacttt aaattctaca ctgggcagac tccaagattc tgatgggaat ttggagacac 3180 tggatagtgg gtgacagaga aagggggaag tcagcggtgg gctccttatc tggggcttgg 3240 aaagtctggg atagggattt accctgcatc cccgtttgca atcaacagag cccctccggc 3300 ttctgttggt tttggggagg atctggcaag tttgcatgga tccccctcag gggaaaggag 3360 aaagcgtcct cggggacttg ctcatccatc acagttgcaa agggtctcag aggaaatttc 3420 atctggggcg gctgtggatg atatggaagg agatggatgg ggcactttcc taagacagat 3480 tcgtctttct ttccccattt caggcgggga agcccccaga gagtctctcc ctaaatatgc 3540 ctctccatgg ctcccctgga gttaggggga tattgagaga aggcagagag gtggagaggg 3600 agagagagag agcaagagcg agagagaggg agacagagag agagagtgtt tcagtactga 3660 gggagatcta caatttgaaa aggggctgtg agtgtggaac ccatgacaga atgtggcagt 3720 aattgactta aactgctgtc ggtttgcacc tcgcgtctct cacttggctc caaaatactg 3780 ccaagggcag gggggcggtg gaggaatctc agccaggaag gtagtttggg tcaaggccct 3840 ctttcttctt tcctccatct cctgggaggc tctttaggta ggtcccttgg ccaccctggc 3900 ttggtactgt tgggcttggg ctctggggcc agccattgat ttgattccca gctgcctctt 3960 aaaggcttgc cctactcagc aaaaatgctg agttttactg ctgttagcat ggcttccaga 4020 ctcggcagct gttactttct caaggtaagc ggcagccgtt ctgctctcag gcagggaggc 4080 taggagaaga caagggctgt gacgctggga cacagcctct tgtctagctc agcacggagg 4140 ggccgctgaa aagccctctt aggggaaaaa agtagaaata tatttggcct aaaaaatgta 4200 cacatatatt ctaggaagat atatatatat atatatatat caatcacaca cacacacaca 4260 cacacacatg tgttcatatt ctggggagag ataaaagcaa acatgtattt gtaggaacct 4320 gccctagtgg gtgggttcta cctgcaggca cttgcagaca taccattctc tgtccagagg 4380 ctgatacctc aggctgaagc cttcacatag ccacagagaa tctccttcag gaaactcatg 4440 tcagggcaga gctccctgga ttatcttggg tagggggtac tagggccaag gaggagaccc 4500 atttttggga actgcctcat tttctctctt tcctaattca cagacgatcg aaactggaag 4560 gaatattaaa gagcagaaag gcaggactct gtggcctaaa gagggaatga gaattcccca 4620 acattcagta gtgggttagt ggcatgccca ggactagaat ctaggcttat caatcagcta 4680 gatcaatacc ctttccactc ctcccagcaa tatacactct aggactgtat attgctccca 4740 cccctatctt gccaacctgc caccatgacc cctaccaaga caatcgcttg tcttccctca 4800 tcaaacttca cattttcaga gatgcatttc agggtttttc tatctggaaa catggccctg 4860 aagagaaaag atccaatgcc tcaatcacta ttcccgaaag acacacacac acacacacac 4920 acacacacac acacacacac acacacacac acacacacgg tggtgggggg aggactgaga 4980 gagagacaga gagagaacat 5000 <210> 2 <211> 5000 <212> DNA <213> Homo sapiens <400> 2 agaacaactc tgaagcatca tcccaccttc agaatcctcc ctgcagggat gcctgaggct 60 tggtcaattt ctccctctgc tcaagcttgc ctccctccat tccctccctt tatttcctcc 120 cacaaatgtt gactacaaga gcacttccta aaaagcctcc ctctgcctgc taaacaccat 180 ctcagaatct gcttctagga ggacccaacc tgtgcagaaa ttttatgagc aatttgcctg 240 tccattaaaa aatacagcat agactttttc caggttagct ggagatggag tttttttgta 300 tcaaaaaaag agagctaaac tgagaagaga agcaaaggta gaagaataag gttagactct 360 ccctgtataa ctgaatttgg gattgggaat gatatcttgc ttctctatga aatttagaaa 420 ctgtcttttg aagtcttcat ccagtcacat ccaagagtgg tccaggaaga aaaaggaaaa 480 taagaatgaa aaaaaaacaa aaaacaaacc ttaaaattgg atacttcttt gtgccttgca 540 ttaagcctct atgttactgc ccttgtccgt ataagcaact ctagataaaa tcctttctta 600 gaccatctaa tttatctccc cgtgccattg tgaaaatcgg gcaggaatta ctgcgggaac 660 cagtgactga atgggcagtg gtgtctcgca gctgtaactc agagtttaca agggaaggat 720 gaacagcagc catccttcta aaggcattct tcctctcctc cagcacagct cagtctgtgc 780 atgcctcacc aaggagaatg agggaaggcc ttgttgactg cccagcccag agtgtgagga 840 acagcagttg acatgggcgg ctggacggga tagaaaccag catgaacatt tagtgacacc 900 taaggaagag gtgaaaatgg gggagaaagc caagaaccat cagacagctt ctttctgttc 960 ttgccttgga tcttagggga acgtatgtgt tcaagggtgc cttcctccgc agggcctccc 1020 atcccttggc acaaacttcc aaacgatgag tagacatgga atgggcttct tctgaagact 1080 gggtcaccat ccaactcagc cttcggagta ggcggtgggg ctgggggaag aggacggaac 1140 catgaggtct ctccatcact tcccgggtgt gttgagaggc aagcaaatgc ggtgggtggg 1200 cctgggctgt agggcccctg ccatttcggg aggggcctcc tggtgtttag ctctgggatg 1260 tgtgaaaatg tgttggtgaa aagcgagagg cttacacagc ccttcagggg aagaggggct 1320 ggggcgctgg gggcggcgtc gggatgagtg cagaagagac gaggcctctg gacagcggag 1380 gaggagggga gggcgccgag gcgcggtgcc agctgccgcg cacaggggcc ccgcggcgga 1440 gccgagccgc gggcacgctc tgccctgtcg ggagagctcc gggagcggcg ggaggggcgg 1500 aggggcgcag tggggcccgg gcggctgcgg ccgcggagcc ggggcacctg aggaggaagg 1560 agggtgggag cgagggaggg aggggacggg cgcagaccga aagtggggaa agaaggtgca 1620 ggcaggcggg caggcgggcg ggcgccctgg cccagggccg cgggtgcggg agcccggcga 1680 ggtcgagctg ggcggcggcg ggggccgcgc cgagggagga ggggaaggcg gaggcgcggg 1740 gagcgtgttt ggggcgccgc ggcggggagg gtggcggccg ctggtgcgcg cggggcgctg 1800 tgtatgcgcg ctcccccgct cggggaggaa gatggcccaa aagggaaagt tggggtgacg 1860 cgcgcggtcc ccggaggctc ggcggggggc accgcggcca gcccgacgga gcggcggaca 1920 cacaggccgg ggggcgcgca gtccgggcgc cgccgcggcc gccccctcac tgcaggtggc 1980 agcgggtgcg ctgggtcccg gcggccgcgg gcgcgggcgg gcgcgcgggg gagcccggcc 2040 gagggatggg ctgcgccccc agcatccatg tctcgcagag cggcgtgatc tactgccggg 2100 actcggacga gtccagctcg ccccgccaga ccaccagcgt gtcgcagggc ccggcggcac 2160 ccctgcccgg cctcttcgtc cagaccgacg ccgccgacgc catccccccg agccgcgcgt 2220 cgggaccccc cagcgtagcc cgcgtccgca gggcccgcac cgagctgggc agcggtagca 2280 gcgcgggttc cgcagccccc gccgcgacca ccagcagggg ccggaggcgc cactgctgca 2340 gcagcgccga ggccgagact cagacctgct acaccagcgt gaaggtaaat gccccgcgct 2400 ggcacacgcc gtgggggccg tccgccccgt cggcggggct cgcacgggta gggggctccg 2460 gcggagttgg gtgaccgtga ggcggttggt ttggagaggt tgtcactaag gaggagttta 2520 cttttcattt gtggagatga tgggagccca ggaaatgtgg tcagaaaaag gcccctggag 2580 gggtcctgga agcgtcctta gctggtcctg ggggactggg cggggaaggg agcgcagaag 2640 gaagcaggtg ggctggcctg ttcctccttg agggcaggaa ggctgtggct tggtttatgc 2700 aggaagaggg gtggggacca ttgagagcat tcggtggcca gtcctgttga atgaaatctg 2760 agcactgagc tggatttgcg tgccttgtag gtgactggtg cagttgcagc accaggatag 2820 atagtgcccc atattccgat ttttacctgg gattaccagc caggctggag tctcagcaca 2880 ggaaccgagc gtagggattt gtgaatgaat gagtgttcgt gttttaagag atgtgggaac 2940 ggagcagagt ggaacctgtt gtttgtcact gtaacgtttc tctgggttgg ctgcatccta 3000 gacagaattg agagaaccgg gccatgagtt cggagtgtca gcagagccac cgtgagggga 3060 cgtggtttcc agtgcagtac agctgtctga ggatgattct gcacatacaa ctgatcttcc 3120 cagagagtgg gattttgagg aagtggaaaa aattgtttag atggttaaag cagtcgctaa 3180 atatttattt cttaaagaca gaagaaaaat aattatttaa atagtgtcct cccgtatgtt 3240 ctcaaagtac cgttaaatcc aagaggcttt tcattgtgta aatctgggca ctgggtcttt 3300 tttcctttca gcaaacaaga taacaatggc atatcctatt gtacagagag aaaaaaatac 3360 tcctaatgtc agatagaatc acagccttta cctggtcaat aggttaccaa gaaccattcc 3420 atggattatt tgtcaaagac acatttgtat ttttaatagt taaaaacttg gtcttcattg 3480 gatgaaggca gcccacagtg gaggagtgag gagaaggata acatgttttg gcttcaatct 3540 ggaagtccaa aagtttttta atgaacctat ttttttttta acagcatctg attgtttaac 3600 atgaagcttg actgatgttt gcttgtggtt gcagtgtttc tctggcagta atcataatca 3660 ccattatagt aaaaacatcg tttattgagc acctactatg tgccaggagt aagctttctc 3720 tcctaactat tgaatgttaa cagtgtgatc ttagcatagc atgtctgaag actagagtct 3780 aggatttatg acagtacaaa cctaccagtt gcccattcgt tacagaggca tgtgctgaac 3840 gctatacttt gcttttgtca agctctctct ctctccccct ccctgttcac acaaagttag 3900 ctttaggagc aattttcagg gatgaaaatg ttaaatttgg tgaaaatatt aagttgggat 3960 attatcagta aagaccatcc ttttgcctga cggttgaaga cctgaagcag tagttcaaaa 4020 ttgtgacagc tcatgaactt tgtgatactt tttttggttg ctccagagaa attgtgaatt 4080 tttgttttct agaatatcta taatctcagt ttagtacttg gcaactctag ctactccatg 4140 tttgttcagt agaagaaaga atactgaatg aacccattct gtacattttt aatacttccc 4200 tcaagaggct gcttagagac caaatgaagt aaaaactgaa gggacaatga gccccaatta 4260 ttagcagctt gtataaactt gtattaatct cattgctact tggcttaagt attaaaaggc 4320 ttgacctggg taggagttta actcattttg ctttatcaag aagattccca aataagagct 4380 taattcactc taacccatta ctgggaatgt tggaaatgaa acccctggtt cattgactta 4440 acactacttt gcattgtgtg gatctcagaa agtatatatt tgaagtgaaa tttgagacca 4500 gagttattta tgcaaatctt acagaattat tatcacagaa ctgccctaat atatttaata 4560 gtatcctttt aggcctgtga ctcggtgttg tattgtcttt tctcaatcgt aaagttcaca 4620 tgttctgtgc ccttttgttg ctgagataat tttataaaaa ctttacattc atttttttgt 4680 agactaagca ttcttttttt gccgtaagag tttgagatag ttgctaggca cgtggtggtg 4740 ccccacccct ttccaaagca cacaggtgga aaaataaaat ctttaccatt tggtcctgtt 4800 tacagtggtg gaaggttgta atagtttcca ccccctcccc ctggccattt tacatcaatt 4860 atgaagtgta tcctcattat ctacttggcc tctggtattc cccagagtgc aaaaatgtgt 4920 gcttgatctc tctctctctc ttttctagca ataattcatc tcattaagct tttagaatga 4980 tgaggcatta tgatgtcaaa 5000 <210> 3 <211> 5000 <212> DNA <213> Homo sapiens <400> 3 aagaaaaata gacctattgt aagtgggata caattatttt tccctaacct ccagccctgc 60 agcatttcta gtaacagatg ttagatgaga agagttattc ttctaagctt tacagaatgc 120 taaataaatt atctcaaagg ttgactcagt aagaatttca aagagttaat tgtcagtatt 180 gaactttgtc aactatcatt ttcatttatt catttttgag tatctatttt gcatgaggca 240 ttgctctagg gagttgggac atatcaataa agtcatcaaa gttcttcaac attggagctc 300 aaattctgtt ttgcgggggg gagtagggaa atagatacta aacaatagat tttataagta 360 attattttgg tttgttagaa agtgatacgg gaaaagcaaa ctggaggaca gtgaatcagg 420 aataggagaa gaggttgcag cttagagtgg tttgggatga aatgatagtg ccacagaaat 480 ggagtgtgcc acacgcatgg gggagtaggg ggtggagcac gttgctagag aggggctaga 540 catggagata tgtctctatt ttcagcagag aaggcctgtc aagatattaa aagtacatct 600 cattgtcccc ttctcccact tgttggagtg cttcctggcc ttgtctctca gagctgaaga 660 tgacataaat cataaaagaa ataaatggta ccgcctgggc tctctgatct tataaggtcc 720 cacctaattt aagctctggt ggatagccta tttcccatta gttgtctcaa ttcacagagg 780 atggagaaag aaagcactga atgagatgtg cagctcaact caatgggcac ttgttagccc 840 caattatttg gctaggacaa ctttattcaa gttaatggaa tacaaaactc agcagttctt 900 atggagactt caatgtgctg tagaacaaac cacagataaa tgaactgtct gaaagaataa 960 tatgaggcac ggctaaacaa gtgttgtgga agagaaagat cactgtaatc tagagttagt 1020 tagagttagg gctttctgga aaagtgagac caacattaca ccttcgaaaa ctagtgtggt 1080 ggtggtgcaa gtggaggaaa atgggaaaga ggcaacagtg tccataaaga cacagaggca 1140 gaaagggcag aggccctggg atagttaaaa gagaagtcta gctgagggac ttcttgactt 1200 gactagcagc aattagccat gactaataag gctttccaca ctccaaagac ttagatggag 1260 ggataaaaaa ccatctaatg gcagactgtg gtagcctccc tagagacaca gagctgggcc 1320 ggatgagtcc aggcactgac gtgatccatt atctttcacc ttaaagagta aaagggaaac 1380 taaagttaat tacctccacg aaacaaaaag gtgccttctt gtgcttcaat tacatggata 1440 tattctacta gtctaaaagt atcttctcac ttctttctgt cactgtgagg acttgagtca 1500 gaagaaagtt taaatacagt cattgagctg gaaagagtgg aaagagaagc aaagaggggg 1560 aagctgtagg aaggacgaag tcacccccaa gatacatggt tactgcttac accaagcaag 1620 ctgccttggg aacgcttccc ccgagcagcc agaatgctca gcagtggaag acacctctat 1680 tcctgtaggc gagtcctggg aagctggtca atctgcaaat gccaattccc agcagtgagc 1740 tcggtccacg tgtaaatcaa gatttgggga aagagtaggg tgggtggcat ggttgacaat 1800 gtcatcagct ccctcctctg actcctgtgg tcgtgccccc atctactctc actcagctac 1860 accccacctt cggatttgtg atggacgctg ggtccctagt aaccacagca agtgtctccc 1920 ccgcacttcc cccttcccca cccccacccc cacccccaac caccacccca gcgatggagc 1980 ctactctgct ccaagccgcc gctaagaccc ggagaagcgg aatttcactt tgaaattccc 2040 ttgcctcgtg agggccggcg ctgggcatgc tcagtagccg cggcgctgct gctgggctgc 2100 tgggctggcg cggagtccac cctgccgtct ccgccttggc ttctgggcgt ccagaaggcc 2160 aggcatttgc cgcctctgag cgcttctgtt ccccttaccc gcaacctcct actgctcttc 2220 ctctctccct ctcttaggga ggttgaagct ggtgctggtt tctgtcggcg ccacagactg 2280 actgctctgc aaaccccagc cgaggacctg aatcccggag actagaagac ccttggcggt 2340 ggctctttct aatagcactt tacctgaagt ggggtcgtgg tggagtttct cctccacctc 2400 tcaatgcaaa cactatgcgg agagcagtct gcttccctgc gctgtgcctg ctccttaatc 2460 ttcacgctgc aggtaagggg ttgccaggct tagagccgga gctgtgcatg agatgggaaa 2520 ctgcacatgc ttaaggactc taggaaaagc ttgccttgcg aagaaagcct ttctaaaaag 2580 gtaaaacagg acagtacctg acagggaagg ggtgagggag tcgtgcacct gttggaaaac 2640 tgaggccgaa aacttaacct aaaattagct cttgattttt ctttacttta tacgaaaata 2700 gtgaacattt tcaatatgaa tacaggtttg ctcccccctt cgcctcccct ccgtctagat 2760 tccctgctct tgtttctagg ctatgcactt aactgtcaat ttttcaggag agggataaga 2820 catcctgcta gatgtaacct tttctactgc agcggctact acattcataa ggtctcttgg 2880 tctagcgagc gctcatagga aggcattggc tgtaacctga tggaccacat ctccgcccaa 2940 aagatcgaaa tacatggttc acattagtga tgctgcacca ggctcctttg gcccctgctc 3000 ttgtcactcg aattttctaa gccaataaga ggaagaaaag gtttcaaata gaatcctctt 3060 gtctctttca gcaccgtggc tagcgtccgc tggcatatta aaaaaaaaaa aaaaaaaaaa 3120 aaaaaaaaaa acaattacag ttccaaagct gacaaccctg ggttaggcac tgtccctcgg 3180 acagatttga taaggtgtaa ccaagagtaa cagtgagact gctcccaaat ataaatagat 3240 ttgaggcacc agaatcaata tcaagtctcg tgaggaagca aaccttggga agtacctgag 3300 agatattata tgtggtgtcc ctgctttttg catacatagc acctctattt aatgcagaca 3360 gacttgctag gctgaattcc ttatcattgt cacacacaca tgaaccaaaa taaaatatgg 3420 cagggagatt ttagaaaccc tactgtgaca gcagttgtcc atgcagtagt cctgtaacaa 3480 ccagagacga ggcattttcc ctgagtgtgc ttctcatcca atcacacata tccattcatt 3540 ccttcaagca acatttgctg agtgtatact atgttctagg ggtaatgagt aaaaataatt 3600 gttctgaaat tacaatcctt gcagtatctt ttttaaaaga gtgggtgaat tttattctct 3660 gtgtacaata gtaggagtga aatatggtac tttcttattc cagctaacgc tattatttaa 3720 atgtatcatg aatcttttga gcaaataatg cataggatta ttaaagctat tattaaatac 3780 acatgttaat tgttataatt atgatgttat attgtggagt tttcaatagt tctttggcac 3840 attttgagaa gtagaaaata gctatgactt gattattaaa ttatttgcaa acagctgtag 3900 tgaattattt cgttaccaca tacaattctt caatgctgaa atgtgggatg aaaacataca 3960 cattgggttg agttttattt atagtattgc ttaataatag actgacacaa acattcatta 4020 cagaaaaatc tcaattctta ctaaggtagc aatgatttag ctggtctgcc aacaatatca 4080 gtgtagtcat tttttcaaaa ctaacatata aaaaagttta aagatcacat ttgatacatc 4140 ttgagtaatt atatcttaaa agttataaat aaactgaagc atgtgaaaaa catgtcttga 4200 tttactgtag atgaatattc gggatgagaa aaatgaggac ttttgctcat cagatgagaa 4260 acctagaagc ctgtagccct agattttacc tggagttctg ccaaagggta tattaccaaa 4320 gccaatccaa tccatctcta cttcaacttt ttaatttgca aaacaaagat aaaaagtaag 4380 attaatcaac caattttaat ttaataagaa aatgcattta aggacatttt tatatttcaa 4440 ttgcaagata ctattcattg tattcatatt taatatagta aaatacattg gtgttttatc 4500 acagcagatt gtaagtttaa tattatagtt ctcttgccaa tttcatttta aatgcatttt 4560 aattttaaac agcaatttgt gggtcacaag ctgtcatcag attaaaattt atcacagtta 4620 tctaataaca ttgttagggc ttttcatcag aactaaaaca aacacagctc cttggtctaa 4680 gcttctatac atttggagat taagaaaata agtggatggg atgacaaaga tcaagaaatc 4740 aagatattaa tctctacttg ttgaaggtgt gagtttgtta cttttatgga agacttttct 4800 ctcagaacag aggctgactt cataaagaag ggaaatgaat tctatacatt caggcctctt 4860 taactgtaaa gctaattgat ccattttggt tacgcccagg gtatcagcaa ctgctttatg 4920 ctggtggaac tgatgacacc ttataatgct ttagagatgg gaggaatgtg ctaacgggat 4980 gtaagtgttc tttttagcta 5000 <210> 4 <211> 5000 <212> DNA <213> Homo sapiens <400> 4 gacgaccctg aatgatccct gcctcctagt acttatactc ttgtgtagcc tccacctact 60 tagactcaca gctgattttg tgaccaagag tataatggtg tatgacttca gaggcttggt 120 tcagagtcag aagtggccat ttcatcatcc tctaaatttc tctttggcag attaatgaat 180 gtgagaaagt aaaaatggta tagaagaaag ttagcctgag agatggctgg ctttgtacct 240 gtttggtctt tttccttcct ttcctccata agcaaatact gacgcatgta catatagaca 300 cactcaacac agtgggatgt taaagggaaa ctagagccac tcgccccttc ccaagatcag 360 agaacatagg aaagatgatg actttttgca tctaccctct cctcttgaac tcgcaacact 420 gtcttgaacc acaaataagg tcaagtagga taaatctaca actgtctcct caactgtccg 480 ctcacaagtg tgaaattcaa ccatccttgc tgaagttgtc ttttaatatc agtaatcagc 540 aataaccaac ctctgagctt tgcttatatt attaaatcat tttatttgac cctcaggaca 600 aagctgcaag cccagcatta cattcttcct tttatggaca aaaaaaaaaa aacaaaacaa 660 aaccgagata ggcacgaaga aactaataaa tggctgaaat gctatttgaa tccacatctg 720 tctaattcta aaagtgtgct ttccaccaca tcaccgcacc tacaaggaaa atgcagcctc 780 taccctctcc tgaagaatgt gtaaagaggc aatatggtgt gttttggaaa aagcatcttt 840 gcagaaagaa agcattagct tcagtcatat tagcatcctt atccagcagc agtagcatta 900 ttttacatat cagttactcc tccccaacaa ctcaatgaat tagatgccac aatcatcctc 960 actttcaaat gtggagaccg aggcacatct ctaagactgg agttcaggca ttctggctcc 1020 agtcttagag atggttaagg gttcacactc ttaaccattt attacaccat agagctcacc 1080 aggtttgagg gaaacaggat caaatcaaaa gagtcactca ggactccagt cctcactcaa 1140 ggacaaactg ttccacctcg gacagggaga gtttccgcat tctgagaccc agcataacag 1200 gtcctgaccg gcatctggca ctcggactcc caatcatact ggatcacact ggctcgggat 1260 gtgtaaagtc cagggcttct cacatttgat gacaccaaag ccgcctaaaa acaagagaga 1320 attaacaact acctacggcg gtctgatatt tgcccaagag atgccgcccc ataaaactcc 1380 tttacatctt tataacgttt ttattttgcg ttctccttca taacccacat ttaactcacc 1440 atagatgtaa tgtttaaaat tagttaccag ataaactctt acgcttccaa actttaaggt 1500 tccttcgaaa ccttctggta aaactgttgt tccacggaaa tgggaacgta acggatgagg 1560 caatcttcca cagccgcaca cagttgtgta tccaccgcta aacggtccca gtcatacatt 1620 caacgaccca cgcggagtca gaagctacca ccacacactg tcaaaatcac gcacacacag 1680 tgacggcccc ttgcccactc ggtcactcgc ccacaatctc tcgctagaga atcacacgca 1740 gatagcacac ccagcaccac agaccccagg aagcaaccca gggactcgaa cacacgaaca 1800 gcactcctcc gcgcactgcg caggcacgcc tgcgtccggc tcaccctgaa acatcgcgag 1860 atccggcttc aaggccgggc tgctgccttt acgcctaaag actatgtttc ccggaagaca 1920 ctgcggcgcc ggccctatca tggcgcagca tcggtgtgct ttgtgcgtct gcgccatctt 1980 ccggctgcgc acggcgaatc caccggtacc gtggtggaag cgcgccctgg gctgccgggg 2040 gcgcggccgc ggtggcactt ggacccgagg aggcggcagg tgagaggttc cggagctttc 2100 caggcgctct ggggtccagc aggagctggt gcccgggccg gttgggtctc aggcctgaga 2160 agaacgcaga cgtctcgcct catcgtcgct ctgtggcttt accggcgtga gactacattt 2220 cccgccggcc ctcgcggcgt gcgctttctg gcgccccctt tctgcttcca gcctcatagc 2280 ccaggtgcat ggacccctta ggtgggtgcg caggggtctc cgaccccctg aaattgcgga 2340 tgttttgcat tcactgttat gcgtgccttt tttttttttt aatctgagag gaaatcttgt 2400 ttcatgaggt tctcagagag ttacaagacc ccagaagact tagaaccgct agtttagaaa 2460 aacttatact tgggaacatt ttatatgttc aaattctatg tccagcaatg gggaaacact 2520 tgagtggatc acaggccatc ctttgtaaat aggatatcgt gcagttaata caaataatgc 2580 ttataaaagt ggtcataata aggaaaagct gcctattaca tcatattaag caacattcat 2640 aattatttca cttacacgtt acttagaacc aaatgtttgt aatgcagtag gcaccttata 2700 tactccttta cagagtactt cttgtttaac agtgagctca agaactggtt aaatgacaac 2760 actgcgaata cttgggtgtt ttttcttcca atttttttgt tttgttttgt ttttagcctg 2820 tgtgttagtg agtttgactt ctttttgctg aatgttctca aaggattcag tagttaatat 2880 ctttgcttgt cagtttttga tagtcatttt tgatagtcac ttggcatccc attatatggc 2940 tgtgccaaaa atgattcaac ctcttggcta cttgtggact tgcctactta cgtactaatt 3000 ttcagttctc ctatttagca aatattattg gacatgcagc tttgtgcact gctgtgtctc 3060 tatggaagga cacatttatg tgctgataat ggagcactgg gcgtgctgtg tgtaatttgt 3120 ctcttactgt tccttccaac tctatcccat ttgttgatgc aaggcatgct ccatgaagtg 3180 gcatgcacaa agcctcagtt tgggctgtag agctttttgt atatttggcg gttgggaaaa 3240 gagccaggta gactttcaaa ggtgttatat ttgctgcagg agagagttaa atggcctagt 3300 cctggaaagg ctttattcta ggctggggct atgcaaatga tgcaggcctt ctatgctccc 3360 tggcttgagg gaagccctcc ttgtccttga ggtaacatgt gggtctcagt cactagaaca 3420 gccctcagca tccctgtctt agacttgcat ttgcagcaaa acagaaccac tggatgttta 3480 ggggaaatgg gagggaaagg accactggtt cccctttgcc ttgcagcagc taaagctggt 3540 ttcctggggt tgggtgagga tccctttgcc tccccctcct tctcattttc ttggtgggca 3600 gagggcaagc aggtggggac ttgtaacttg gcattctagg aactgtgagg caagcctagc 3660 ctctcatgtt tgctcctcc tcccccagct ctaccgtcgc aggactctgc ccttccccaa 3720 gagaggaacc agaaggagga cctatcagcc cttgaaattc taaaagtcat gtcccttgtg 3780 agtttttaaa tcccataaat atttgctttc cttatcttga aactttccac ctgggttcca 3840 tccagaaatt tggttctcag actttcccat tttagggaat aaaaggacca ggcaggagtc 3900 tcctctccca ttgtcctctc ccctctgaca aatgcccaca gatttattct ctatagcatt 3960 ctgcatctcc tgatggtttt cgtctgctag gaggagaaac tcatttccct gtatgaaatg 4020 agagcatcct ttgtaagaac tgaagtttga agagatggaa tgaatgctta ggagcaatca 4080 catatagaaa agcaggggag gtggtgttgg ttggtttcag tttcagcgca tggtacagtg 4140 tcctgctttg ggaatgatta atgattggat atgtaattga ggtttggctg ggaatccatc 4200 aaaaatttga tgccagaatt aaagtctagt ttgacgttgg acagagcatt tgaatctggg 4260 acatcctcca ttagtttctt tagcttaaga ggaagaaaag tacagagctg aggatggtga 4320 gcgatgtagc aagtgagatg atcatctagg tgggtgagtg gtcagggctt ggaaaagcct 4380 tagagatctg atccaacctc gggatccaaa ataagaggat gtttgagtca gttgtttcac 4440 ttgatagaaa attgtgcact caataaaagt tatctccatg gtttttccca tgaagaaaaa 4500 tgcttgtgtt atgttccaac ataatgatga actttcatct ccaagaaatt cttgacttac 4560 atgaaaatcc tgtatctaag atagaaaaca tgtttccata attttaaaaa ataaaatatt 4620 tggggaaaat ccatcagaaa tattttttgt tgttattgtt attccaaaag atagaagtga 4680 taggtctcac cttgagaatt tattgtatgc caagtataga gtgggcagtg tgctttatta 4740 cactgtttct taacaataag gaaaactcca tttagcttga ttttaatttt catcccagac 4800 ctgtacacag agcattgatt tgcagttaaa aggaatgttt gagaacaatt tgatcattct 4860 gttttactca tccccatttc ttctgtcacc tttcacattc agtcccaccc ttcttgttca 4920 acaaaacaac cccccctccc gcaaagacct gccccatctc ctttcatccc actgtgaacc 4980 attgaaatac atatatatca 5000 <210> 5 <211> 27 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 5 gttttcgtcg tttttcgttc ggagatc 27 <210> 6 <211> 25 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 6 gctctcgctc tcgctattac tcgct 25 <210> 7 <211> 23 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 7 tgtgtatgcg cgttttttcg ttc 23 <210> 8 <211> 23 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 8 acctatatat ccgccgctcc gtc 23 <210> 9 <211> 24 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 9 cggcgttggg tatgtttagt agtc 24 <210> 10 <211> 29 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 10 aattacgaat aaaaaaaaca aaaacgctc 29 <210> 11 <211> 28 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 11 tgacggtttt ttgtttattc ggttattc 28 <210> 12 <211> 21 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 12 cgaacgcaaa cgtacctacg c 21 <210> 13 <211> 26 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 13 ggttaagttt ttttttyggy gtagtt 26 <210> 14 <211> 26 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 14 tactccctct acctcccrac tctctc 26 <210> 15 <211> 28 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 15 ttgtggygta gaggattatt agtttggt 28 <210> 16 <211> 22 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 16 ctaaaaacrc aacccatccc tc 22 <210> 17 <211> 27 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 17 ggaattttat tttgaaattt ttttgtt 27 <210> 18 <211> 33 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 18 ccccacttca aataaaatac tattaaaaaa aac 33 <210> 19 <211> 30 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 19 tagtgayggt tttttgttta ttyggttatt 30 <210> 20 <211> 26 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 20 taaacrtaaa aacaacaacc cracct 26 <210> 21 <211> 5000 <212> DNA <213> unknown <220> <223> bisulfite sequencing of DBC1 <400> 21 ggtttttatt tgaagttgtt tgattaygtt gttttagatt gattttagaa ttgtattatt 60 agtagggtag ttgttgttta ttaatagtta tggaagtaga ttttagttgg gggatgatgg 120 ggaaataaag agaaagatag ggaygaataa aggaaggaat ggggtataga tagaaagaga 180 gagagagaaa agaaagaaag gaggagaggt aagagatagt gatttggggt tttatatygy 240 ggtgggtggg agaaggagtg tgtgtggtgg gygtattttt tygtggttag aattaaagtt 300 agaattgatt tggggaattt gtgtttaaga tttgaatttg gattagaagt taagggaaag 360 agaaagttgt ttaagaaaaa agggagaaat ttttattagg ygaaaataga aatygttgat 420 tgggtttgtg gttggagagt tgttygtggt gttgattttt ttttattggg attttagtga 480 aggttgagaa gttttgtatt ggttgttttt tttttttttt tttttttttt ttagttttga 540 ttttttgaga tttaggatta ttttggttag ttygatgttt tttttttttt tttttttttt 600 ttttttgtyg attaaataat ttttttygag tttttagttt attatttaaa tatttttttt 660 ttattttgtt ttaagatttt ttttttttta attagtttat tggtttttag attgttttag 720 gtttttttgg tggttatatt ttttttttat tattttttga attaaaaaat aaataaaaaa 780 tatataatgg taattataaa agtagataat atgtattgag tatttattag gtgttaggga 840 gtagtttaag tgtttttgta tatattaatt tatgagtagt ttaagtgttt ttgtatatat 900 taatttatta taattttata ttaattttat tagatgggta gatatattat tattttatta 960 aatagataag gaattgaggt ttaaggggta agtgatttta ttaagattag atagttatag 1020 gattttaatt tttttggagt ttgtattttt attgaatttg gtttttttat atattttaat 1080 attttttttt atgatttttt gtttttttat ttggtagttt taatgttttt tatttttgtt 1140 taatttttag gggattaggg atttgtattg ttgatttggt ttaaattaga agggaagtaa 1200 agtaaataaa tatattaaat gtgygttgtg tgggaattat tataygaggg ttttattttt 1260 tgttttagga agaggtttta tgttagtagt tttagtttgt atttaggttg attgtagagt 1320 attttgtttt ttatttttat gttttagttt ttgtatttty gttttttttt ygtttttggt 1380 ggtttttaga gaatttygtg tttttttagt tttttttttt tatattttgt ttaygtagag 1440 aagtttttgt tttattttgg gaggttaygt gggttttygt ttatatatyg agagaaataa 1500 atagtgttaa atatttatag agagaygygt agatataaay ggatttatay gggtaatttt 1560 ygagataaaa tttatattyg atggatttay gyggtygtgg aaatatttgt ygttttagaa 1620 atatttaggt attygygata tatatagtat agttaygttt aagggtatta ggatttyggg 1680 tttgygygta tgygyggttt ttttggatgt tygtgygtat agatataata ttttataygt 1740 tttagattta ygaaattttt tayggtttag ttttagttta ttygggtygt ttttttttyg 1800 aggyggtttt tygttttttt ttttttygtt tttttttttt tttygtttaa agatgtataa 1860 aatattttty ggaagtaatt tyggygttta gttttttttt tttygttttt yggtygtygt 1920 ttttttattt attttyggty gtygtyggtt aagttttttt ttyggygtag ttyggtttty 1980 gtygttttty gttyggagat ygyggygtat ttggattttt ttttttatty gttagtygtt 2040 tygttttygg attttayggt tgtaaattga tttggygygy ggggaggagg agagygtagg 2100 ygagygaatt ygygagagag ggagagagyg agygagtaat agygagagyg agagygagag 2160 agtygggagg tagagggagt agtgatygtt tttyggagty gggatttatg tttgttttyg 2220 ggattagyga aggggatttt ayggttgagt atgagttagg ttgttaggag ttaggtattt 2280 ttaygtttgt agttttygyg tygtgttygg aatgygagtt gtaygtaggg tttttttaag 2340 tttttatyga gtygaataaa aagygttttt tygtagtttt tygttaaaga yggatattga 2400 ttttaggtaa ggyggygtyg ggtgtagygt ttygtagttt ygttgttttt ggattyggtt 2460 tygggtygta ttyggggygt tttygygttt ttttgttttt ttttttatyg gtatttttgt 2520 tygtttttta tttggtygtt tygtygtttt taagtttttt ttagttttag ggagggggtt 2580 tttgtgtttg gggtttaaag ggttaattgy gggtttgagt gagtggygtg tgtgttttyg 2640 ygygttttyg aygtgtgtat tatggtggga atttgatgtg gtgttagtgt gtttgygtgt 2700 gyggygtygt ttgygtttga tgygtgtgtt ygtggtgtgt gtgtgtgtty gtgtgtgtaa 2760 gggaggggtg aagagagaga ggttttataa tttatttayg gygygatgtg tgtgtgtatg 2820 tttgtaygta tgtgtttgta tgtgtgtttt ygtgtgtttt tttaattagg tttttttagt 2880 ttatayggaa tgggattttt attataggat taygtagtta tygggaaatt ygttgtggat 2940 ttttttttgg ggttttgggt ttggggtttg gggaggatta tggggttgta gatggtattt 3000 tatttagttt aatgttggta ygtttgaagg aaaatttttt taaayggtgg aattttgtta 3060 tattgggatt tatagtttaa tatgaaagag atatggttaa ttttygaggt aaatgagayg 3120 ttgttatttt aaattttata ttgggtagat tttaagattt tgatgggaat ttggagatat 3180 tggatagtgg gtgatagaga aagggggaag ttagyggtgg gttttttatt tggggtttgg 3240 aaagtttggg atagggattt attttgtatt ttygtttgta attaatagag ttttttyggt 3300 ttttgttggt tttggggagg atttggtaag tttgtatgga ttttttttag gggaaaggag 3360 aaagygtttt yggggatttg tttatttatt atagttgtaa agggttttag aggaaatttt 3420 atttggggyg gttgtggatg atatggaagg agatggatgg ggtatttttt taagatagat 3480 tygttttttt ttttttattt taggygggga agtttttaga gagttttttt ttaaatatgt 3540 ttttttatgg tttttttgga gttaggggga tattgagaga aggtagagag gtggagaggg 3600 agagagagag agtaagagyg agagagaggg agatagagag agagagtgtt ttagtattga 3660 gggagattta taatttgaaa aggggttgtg agtgtggaat ttatgataga atgtggtagt 3720 aattgattta aattgttgty ggtttgtatt tygygttttt tatttggttt taaaatattg 3780 ttaagggtag gggggyggtg gaggaatttt agttaggaag gtagtttggg ttaaggtttt 3840 tttttttttt ttttttattt tttgggaggt tttttaggta ggttttttgg ttattttggt 3900 ttggtattgt tgggtttggg ttttggggtt agttattgat ttgattttta gttgtttttt 3960 aaaggtttgt tttatttagt aaaaatgttg agttttattg ttgttagtat ggtttttaga 4020 ttyggtagtt gttatttttt taaggtaagy ggtagtygtt ttgtttttag gtagggaggt 4080 taggagaaga taagggttgt gaygttggga tatagttttt tgtttagttt agtayggagg 4140 ggtygttgaa aagttttttt aggggaaaaa agtagaaata tatttggttt aaaaaatgta 4200 tatatatatt ttaggaagat atatatatat atatatatat taattatata tatatatata 4260 tatatatatg tgtttatatt ttggggagag ataaaagtaa atatgtattt gtaggaattt 4320 gttttagtgg gtgggtttta tttgtaggta tttgtagata tattattttt tgtttagagg 4380 ttgatatttt aggttgaagt ttttatatag ttatagagaa ttttttttag gaaatttatg 4440 ttagggtaga gttttttgga ttattttggg tagggggtat tagggttaag gaggagattt 4500 atttttggga attgttttat tttttttttt ttttaattta tagaygatyg aaattggaag 4560 gaatattaaa gagtagaaag gtaggatttt gtggtttaaa gagggaatga gaatttttta 4620 atatttagta gtgggttagt ggtatgttta ggattagaat ttaggtttat taattagtta 4680 gattaatatt ttttttattt tttttagtaa tatatatttt aggattgtat attgttttta 4740 tttttatttt gttaatttgt tattatgatt tttattaaga taatygtttg ttttttttta 4800 ttaaatttta tatttttaga gatgtatttt agggtttttt tatttggaaa tatggttttg 4860 aagagaaaag atttaatgtt ttaattatta ttttygaaag atatatatat atatatatat 4920 atatatatat atatatatat atatatatat atatataygg tggtgggggg aggattgaga 4980 gagagataga gagagaatat 5000 <210> 22 <211> 5000 <212> DNA <213> unknown <220> <223> bisulfite sequencing of PDE8B <400> 22 agaataattt tgaagtatta ttttattttt agaatttttt ttgtagggat gtttgaggtt 60 tggttaattt ttttttttgt ttaagtttgt ttttttttat tttttttttt tatttttttt 120 tataaatgtt gattataaga gtatttttta aaaagttttt ttttgtttgt taaatattat 180 tttagaattt gtttttagga ggatttaatt tgtgtagaaa ttttatgagt aatttgtttg 240 tttattaaaa aatatagtat agattttttt taggttagtt ggagatggag tttttttgta 300 ttaaaaaaag agagttaaat tgagaagaga agtaaaggta gaagaataag gttagatttt 360 ttttgtataa ttgaatttgg gattgggaat gatattttgt ttttttatga aatttagaaa 420 ttgttttttg aagtttttat ttagttatat ttaagagtgg tttaggaaga aaaaggaaaa 480 taagaatgaa aaaaaaataa aaaataaatt ttaaaattgg atattttttt gtgttttgta 540 ttaagttttt atgttattgt ttttgttygt ataagtaatt ttagataaaa ttttttttta 600 gattatttaa tttatttttt ygtgttattg tgaaaatygg gtaggaatta ttgygggaat 660 tagtgattga atgggtagtg gtgtttygta gttgtaattt agagtttata agggaaggat 720 gaatagtagt tattttttta aaggtatttt tttttttttt tagtatagtt tagtttgtgt 780 atgttttatt aaggagaatg agggaaggtt ttgttgattg tttagtttag agtgtgagga 840 atagtagttg atatgggygg ttggayggga tagaaattag tatgaatatt tagtgatatt 900 taaggaagag gtgaaaatgg gggagaaagt taagaattat tagatagttt ttttttgttt 960 ttgttttgga ttttagggga aygtatgtgt ttaagggtgt tttttttygt agggtttttt 1020 attttttggt ataaattttt aaaygatgag tagatatgga atgggttttt tttgaagatt 1080 gggttattat ttaatttagt tttyggagta ggyggtgggg ttgggggaag aggayggaat 1140 tatgaggttt ttttattatt tttygggtgt gttgagaggt aagtaaatgy ggtgggtggg 1200 tttgggttgt agggtttttg ttatttyggg aggggttttt tggtgtttag ttttgggatg 1260 tgtgaaaatg tgttggtgaa aagygagagg tttatatagt tttttagggg aagaggggtt 1320 ggggygttgg gggyggygty gggatgagtg tagaagagay gaggtttttg gatagyggag 1380 gaggagggga gggygtygag gygyggtgtt agttgtygyg tataggggtt tygyggygga 1440 gtygagtygy gggtaygttt tgttttgtyg ggagagttty gggagyggyg ggaggggygg 1500 aggggygtag tggggttygg gyggttgygg tygyggagty ggggtatttg aggaggaagg 1560 agggtgggag ygagggaggg aggggayggg ygtagatyga aagtggggaa agaaggtgta 1620 ggtaggyggg taggygggyg ggygttttgg tttagggtyg ygggtgyggg agttyggyga 1680 ggtygagttg ggyggyggyg ggggtygygt ygagggagga ggggaaggyg gaggygyggg 1740 gagygtgttt ggggygtygy ggyggggagg gtggyggtyg ttggtgygyg yggggygttg 1800 tgtatgygyg ttttttygtt yggggaggaa gatggtttaa aagggaaagt tggggtgayg 1860 ygygyggttt tyggaggtty ggyggggggt atygyggtta gttygaygga gyggyggata 1920 tataggtygg ggggygygta gttygggygt ygtygyggty gtttttttat tgtaggtggt 1980 agygggtgyg ttgggtttyg gyggtygygg gygygggygg gygygygggg gagttyggty 2040 gagggatggg ttgygttttt agtatttatg tttygtagag yggygtgatt tattgtyggg 2100 attyggayga gtttagttyg tttygttaga ttattagygt gtygtagggt tyggyggtat 2160 ttttgttygg ttttttygtt tagatygayg tygtygaygt tattttttyg agtygygygt 2220 ygggattttt tagygtagtt ygygttygta gggttygtat ygagttgggt agyggtagta 2280 gygygggttt ygtagtttty gtygygatta ttagtagggg tyggaggygt tattgttgta 2340 gtagygtyga ggtygagatt tagatttgtt atattagygt gaaggtaaat gtttygygtt 2400 ggtataygty gtgggggtyg ttygtttygt yggyggggtt ygtaygggta gggggtttyg 2460 gyggagttgg gtgatygtga ggyggttggt ttggagaggt tgttattaag gaggagttta 2520 ttttttattt gtggagatga tgggagttta ggaaatgtgg ttagaaaaag gtttttggag 2580 gggttttgga agygttttta gttggttttg ggggattggg yggggaaggg agygtagaag 2640 gaagtaggtg ggttggtttg tttttttttg agggtaggaa ggttgtggtt tggtttatgt 2700 aggaagaggg gtggggatta ttgagagtat tyggtggtta gttttgttga atgaaatttg 2760 agtattgagt tggatttgyg tgttttgtag gtgattggtg tagttgtagt attaggatag 2820 atagtgtttt atatttygat ttttatttgg gattattagt taggttggag ttttagtata 2880 ggaatygagy gtagggattt gtgaatgaat gagtgttygt gttttaagag atgtgggaay 2940 ggagtagagt ggaatttgtt gtttgttatt gtaaygtttt tttgggttgg ttgtatttta 3000 gatagaattg agagaatygg gttatgagtt yggagtgtta gtagagttat ygtgagggga 3060 ygtggttttt agtgtagtat agttgtttga ggatgatttt gtatatataa ttgatttttt 3120 tagagagtgg gattttgagg aagtggaaaa aattgtttag atggttaaag tagtygttaa 3180 atatttattt tttaaagata gaagaaaaat aattatttaa atagtgtttt ttygtatgtt 3240 tttaaagtat ygttaaattt aagaggtttt ttattgtgta aatttgggta ttgggttttt 3300 ttttttttta gtaaataaga taataatggt atattttatt gtatagagag aaaaaaatat 3360 ttttaatgtt agatagaatt atagttttta tttggttaat aggttattaa gaattatttt 3420 atggattatt tgttaaagat atatttgtat ttttaatagt taaaaatttg gtttttattg 3480 gatgaaggta gtttatagtg gaggagtgag gagaaggata atatgttttg gttttaattt 3540 ggaagtttaa aagtttttta atgaatttat ttttttttta atagtatttg attgtttaat 3600 atgaagtttg attgatgttt gtttgtggtt gtagtgtttt tttggtagta attataatta 3660 ttattatagt aaaaatatyg tttattgagt atttattatg tgttaggagt aagttttttt 3720 ttttaattat tgaatgttaa tagtgtgatt ttagtatagt atgtttgaag attagagttt 3780 aggatttatg atagtataaa tttattagtt gtttattygt tatagaggta tgtgttgaay 3840 gttatatttt gtttttgtta agtttttttt tttttttttt ttttgtttat ataaagttag 3900 ttttaggagt aatttttagg gatgaaaatg ttaaatttgg tgaaaatatt aagttgggat 3960 attattagta aagattattt ttttgtttga yggttgaaga tttgaagtag tagtttaaaa 4020 ttgtgatagt ttatgaattt tgtgatattt tttttggttg ttttagagaa attgtgaatt 4080 tttgtttttt agaatattta taattttagt ttagtatttg gtaattttag ttattttatg 4140 tttgtttagt agaagaaaga atattgaatg aatttatttt gtatattttt aatatttttt 4200 ttaagaggtt gtttagagat taaatgaagt aaaaattgaa gggataatga gttttaatta 4260 ttagtagttt gtataaattt gtattaattt tattgttatt tggtttaagt attaaaaggt 4320 ttgatttggg taggagttta atttattttg ttttattaag aagattttta aataagagtt 4380 taatttattt taatttatta ttgggaatgt tggaaatgaa atttttggtt tattgattta 4440 atattatttt gtattgtgtg gattttagaa agtatatatt tgaagtgaaa tttgagatta 4500 gagttattta tgtaaatttt atagaattat tattatagaa ttgttttaat atatttaata 4560 gtattttttt aggtttgtga ttyggtgttg tattgttttt ttttaatygt aaagtttata 4620 tgttttgtgt ttttttgttg ttgagataat tttataaaaa ttttatattt atttttttgt 4680 agattaagta tttttttttt gtygtaagag tttgagatag ttgttaggta ygtggtggtg 4740 ttttattttt ttttaaagta tataggtgga aaaataaaat ttttattatt tggttttgtt 4800 tatagtggtg gaaggttgta atagttttta tttttttttt ttggttattt tatattaatt 4860 atgaagtgta tttttattat ttatttggtt tttggtattt tttagagtgt aaaaatgtgt 4920 gtttgatttt tttttttttt ttttttagta ataatttatt ttattaagtt tttagaatga 4980 tgaggtatta tgatgttaaa 5000 <210> 23 <211> 5000 <212> DNA <213> unknown <220> <223> bisulfite sequencing of PTPRR <400> 23 aagaaaaata gatttattgt aagtgggata taattatttt tttttaattt ttagttttgt 60 agtattttta gtaatagatg ttagatgaga agagttattt ttttaagttt tatagaatgt 120 taaataaatt attttaaagg ttgatttagt aagaatttta aagagttaat tgttagtatt 180 gaattttgtt aattattatt tttatttatt tatttttgag tatttatttt gtatgaggta 240 ttgttttagg gagttgggat atattaataa agttattaaa gttttttaat attggagttt 300 aaattttgtt ttgygggggg gagtagggaa atagatatta aataatagat tttataagta 360 attattttgg tttgttagaa agtgataygg gaaaagtaaa ttggaggata gtgaattagg 420 aataggagaa gaggttgtag tttagagtgg tttgggatga aatgatagtg ttatagaaat 480 ggagtgtgtt ataygtatgg gggagtaggg ggtggagtay gttgttagag aggggttaga 540 tatggagata tgtttttatt tttagtagag aaggtttgtt aagatattaa aagtatattt 600 tattgttttt tttttttatt tgttggagtg ttttttggtt ttgtttttta gagttgaaga 660 tgatataaat tataaaagaa ataaatggta tygtttgggt tttttgattt tataaggttt 720 tatttaattt aagttttggt ggatagttta ttttttatta gttgttttaa tttatagagg 780 atggagaaag aaagtattga atgagatgtg tagtttaatt taatgggtat ttgttagttt 840 taattatttg gttaggataa ttttatttaa gttaatggaa tataaaattt agtagttttt 900 atggagattt taatgtgttg tagaataaat tatagataaa tgaattgttt gaaagaataa 960 tatgaggtay ggttaaataa gtgttgtgga agagaaagat tattgtaatt tagagttagt 1020 tagagttagg gttttttgga aaagtgagat taatattata ttttygaaaa ttagtgtggt 1080 ggtggtgtaa gtggaggaaa atgggaaaga ggtaatagtg tttataaaga tatagaggta 1140 gaaagggtag aggttttggg atagttaaaa gagaagttta gttgagggat tttttgattt 1200 gattagtagt aattagttat gattaataag gttttttata ttttaaagat ttagatggag 1260 ggataaaaaa ttatttaatg gtagattgtg gtagtttttt tagagatata gagttgggty 1320 ggatgagttt aggtattgay gtgatttatt attttttatt ttaaagagta aaagggaaat 1380 taaagttaat tatttttayg aaataaaaag gtgttttttt gtgttttaat tatatggata 1440 tattttatta gtttaaaagt atttttttat ttttttttgt tattgtgagg atttgagtta 1500 gaagaaagtt taaatatagt tattgagttg gaaagagtgg aaagagaagt aaagaggggg 1560 aagttgtagg aaggaygaag ttatttttaa gatatatggt tattgtttat attaagtaag 1620 ttgttttggg aaygtttttt tygagtagtt agaatgttta gtagtggaag atatttttat 1680 ttttgtaggy gagttttggg aagttggtta atttgtaaat gttaattttt agtagtgagt 1740 tyggtttayg tgtaaattaa gatttgggga aagagtaggg tgggtggtat ggttgataat 1800 gttattagtt tttttttttg atttttgtgg tygtgttttt atttattttt atttagttat 1860 attttatttt yggatttgtg atggaygttg ggtttttagt aattatagta agtgtttttt 1920 tygtattttt ttttttttta tttttatttt tatttttaat tattatttta gygatggagt 1980 ttattttgtt ttaagtygty gttaagatty ggagaagygg aattttattt tgaaattttt 2040 ttgtttygtg agggtyggyg ttgggtatgt ttagtagtyg yggygttgtt gttgggttgt 2100 tgggttggyg yggagtttat tttgtygttt tygttttggt ttttgggygt ttagaaggtt 2160 aggtatttgt ygtttttgag ygtttttgtt ttttttatty gtaatttttt attgtttttt 2220 tttttttttt tttttaggga ggttgaagtt ggtgttggtt tttgtyggyg ttatagattg 2280 attgttttgt aaattttagt ygaggatttg aatttyggag attagaagat ttttggyggt 2340 ggtttttttt aatagtattt tatttgaagt ggggtygtgg tggagttttt tttttatttt 2400 ttaatgtaaa tattatgygg agagtagttt gtttttttgy gttgtgtttg ttttttaatt 2460 tttaygttgt aggtaagggg ttgttaggtt tagagtygga gttgtgtatg agatgggaaa 2520 ttgtatatgt ttaaggattt taggaaaagt ttgttttgyg aagaaagttt ttttaaaaag 2580 gtaaaatagg atagtatttg atagggaagg ggtgagggag tygtgtattt gttggaaaat 2640 tgaggtygaa aatttaattt aaaattagtt tttgattttt ttttatttta taygaaaata 2700 gtgaatattt ttaatatgaa tataggtttg tttttttttt ygtttttttt tygtttagat 2760 tttttgtttt tgtttttagg ttatgtattt aattgttaat tttttaggag agggataaga 2820 tattttgtta gatgtaattt tttttattgt agyggttatt atatttataa ggttttttgg 2880 tttagygagy gtttatagga aggtattggt tgtaatttga tggattatat tttygtttaa 2940 aagatygaaa tatatggttt atattagtga tgttgtatta ggtttttttg gtttttgttt 3000 ttgttattyg aattttttaa gttaataaga ggaagaaaag gttttaaata gaattttttt 3060 gtttttttta gtatygtggt tagygttygt tggtatatta aaaaaaaaaa aaaaaaaaaa 3120 aaaaaaaaaa ataattatag ttttaaagtt gataattttg ggttaggtat tgtttttygg 3180 atagatttga taaggtgtaa ttaagagtaa tagtgagatt gtttttaaat ataaatagat 3240 ttgaggtatt agaattaata ttaagtttyg tgaggaagta aattttggga agtatttgag 3300 agatattata tgtggtgttt ttgttttttg tatatatagt atttttattt aatgtagata 3360 gatttgttag gttgaatttt ttattattgt tatatatata tgaattaaaa taaaatatgg 3420 tagggagatt ttagaaattt tattgtgata gtagttgttt atgtagtagt tttgtaataa 3480 ttagagayga ggtatttttt ttgagtgtgt tttttattta attatatata tttatttatt 3540 tttttaagta atatttgttg agtgtatatt atgttttagg ggtaatgagt aaaaataatt 3600 gttttgaaat tataattttt gtagtatttt ttttaaaaga gtgggtgaat tttatttttt 3660 gtgtataata gtaggagtga aatatggtat ttttttattt tagttaaygt tattatttaa 3720 atgtattatg aattttttga gtaaataatg tataggatta ttaaagttat tattaaatat 3780 atatgttaat tgttataatt atgatgttat attgtggagt ttttaatagt tttttggtat 3840 attttgagaa gtagaaaata gttatgattt gattattaaa ttatttgtaa atagttgtag 3900 tgaattattt ygttattata tataattttt taatgttgaa atgtgggatg aaaatatata 3960 tattgggttg agttttattt atagtattgt ttaataatag attgatataa atatttatta 4020 tagaaaaatt ttaattttta ttaaggtagt aatgatttag ttggtttgtt aataatatta 4080 gtgtagttat ttttttaaaa ttaatatata aaaaagttta aagattatat ttgatatatt 4140 ttgagtaatt atattttaaa agttataaat aaattgaagt atgtgaaaaa tatgttttga 4200 tttattgtag atgaatatty gggatgagaa aaatgaggat ttttgtttat tagatgagaa 4260 atttagaagt ttgtagtttt agattttatt tggagttttg ttaaagggta tattattaaa 4320 gttaatttaa tttattttta ttttaatttt ttaatttgta aaataaagat aaaaagtaag 4380 attaattaat taattttaat ttaataagaa aatgtattta aggatatttt tatattttaa 4440 ttgtaagata ttatttattg tatttatatt taatatagta aaatatattg gtgttttatt 4500 atagtagatt gtaagtttaa tattatagtt tttttgttaa ttttatttta aatgtatttt 4560 aattttaaat agtaatttgt gggttataag ttgttattag attaaaattt attatagtta 4620 tttaataata ttgttagggt tttttattag aattaaaata aatatagttt tttggtttaa 4680 gtttttatat atttggagat taagaaaata agtggatggg atgataaaga ttaagaaatt 4740 aagatattaa tttttatttg ttgaaggtgt gagtttgtta tttttatgga agattttttt 4800 tttagaatag aggttgattt tataaagaag ggaaatgaat tttatatatt taggtttttt 4860 taattgtaaa gttaattgat ttattttggt taygtttagg gtattagtaa ttgttttatg 4920 ttggtggaat tgatgatatt ttataatgtt ttagagatgg gaggaatgtg ttaaygggat 4980 gtaagtgttt tttttagtta 5000 <210> 24 <211> 5000 <212> DNA <213> unknown <220> <223> bisulfite sequencing of ZNF582 <400> 24 gaygattttg aatgattttt gttttttagt atttatattt ttgtgtagtt tttatttatt 60 tagatttata gttgattttg tgattaagag tataatggtg tatgatttta gaggtttggt 120 ttagagttag aagtggttat tttattattt tttaaatttt tttttggtag attaatgaat 180 gtgagaaagt aaaaatggta tagaagaaag ttagtttgag agatggttgg ttttgtattt 240 gtttggtttt tttttttttt tttttttata agtaaatatt gaygtatgta tatatagata 300 tatttaatat agtgggatgt taaagggaaa ttagagttat tygttttttt ttaagattag 360 agaatatagg aaagatgatg attttttgta tttatttttt ttttttgaat tygtaatatt 420 gttttgaatt ataaataagg ttaagtagga taaatttata attgtttttt taattgttyg 480 tttataagtg tgaaatttaa ttatttttgt tgaagttgtt ttttaatatt agtaattagt 540 aataattaat ttttgagttt tgtttatatt attaaattat tttatttgat ttttaggata 600 aagttgtaag tttagtatta tatttttttt tttatggata aaaaaaaaaa aataaaataa 660 aatygagata ggtaygaaga aattaataaa tggttgaaat gttatttgaa tttatatttg 720 tttaatttta aaagtgtgtt ttttattata ttatygtatt tataaggaaa atgtagtttt 780 tatttttttt tgaagaatgt gtaaagaggt aatatggtgt gttttggaaa aagtattttt 840 gtagaaagaa agtattagtt ttagttatat tagtattttt atttagtagt agtagtatta 900 ttttatatat tagttatttt tttttaataa tttaatgaat tagatgttat aattattttt 960 atttttaaat gtggagatyg aggtatattt ttaagattgg agtttaggta ttttggtttt 1020 agttttagag atggttaagg gtttatattt ttaattattt attatattat agagtttatt 1080 aggtttgagg gaaataggat taaattaaaa gagttattta ggattttagt ttttatttaa 1140 ggataaattg ttttatttyg gatagggaga gttttygtat tttgagattt agtataatag 1200 gttttgatyg gtatttggta ttyggatttt taattatatt ggattatatt ggttygggat 1260 gtgtaaagtt tagggttttt tatatttgat gatattaaag tygtttaaaa ataagagaga 1320 attaataatt atttayggyg gtttgatatt tgtttaagag atgtygtttt ataaaatttt 1380 tttatatttt tataaygttt ttattttgyg ttttttttta taatttatat ttaatttatt 1440 atagatgtaa tgtttaaaat tagttattag ataaattttt aygtttttaa attttaaggt 1500 tttttygaaa ttttttggta aaattgttgt tttayggaaa tgggaaygta ayggatgagg 1560 taatttttta tagtygtata tagttgtgta tttatygtta aayggtttta gttatatatt 1620 taaygattta ygyggagtta gaagttatta ttatatattg ttaaaattay gtatatatag 1680 tgayggtttt ttgtttatty ggttattygt ttataatttt tygttagaga attataygta 1740 gatagtatat ttagtattat agattttagg aagtaattta gggattygaa tataygaata 1800 gtatttttty gygtattgyg taggtaygtt tgygttyggt ttattttgaa atatygygag 1860 attyggtttt aaggtygggt tgttgttttt aygtttaaag attatgtttt tyggaagata 1920 ttgyggygty ggttttatta tggygtagta tyggtgtgtt ttgtgygttt gygttatttt 1980 tyggttgygt ayggygaatt tatyggtaty gtggtggaag ygygttttgg gttgtygggg 2040 gygyggtygy ggtggtattt ggattygagg aggyggtagg tgagaggttt yggagttttt 2100 taggygtttt ggggtttagt aggagttggt gttygggtyg gttgggtttt aggtttgaga 2160 agaaygtaga ygtttygttt tatygtygtt ttgtggtttt atyggygtga gattatattt 2220 ttygtyggtt ttygyggygt gygttttttg gygttttttt tttgttttta gttttatagt 2280 ttaggtgtat ggatttttta ggtgggtgyg taggggtttt ygattttttg aaattgygga 2340 tgttttgtat ttattgttat gygtgttttt tttttttttt aatttgagag gaaattttgt 2400 tttatgaggt ttttagagag ttataagatt ttagaagatt tagaatygtt agtttagaaa 2460 aatttatatt tgggaatatt ttatatgttt aaattttatg tttagtaatg gggaaatatt 2520 tgagtggatt ataggttatt ttttgtaaat aggatatygt gtagttaata taaataatgt 2580 ttataaaagt ggttataata aggaaaagtt gtttattata ttatattaag taatatttat 2640 aattatttta tttataygtt atttagaatt aaatgtttgt aatgtagtag gtattttata 2700 tattttttta tagagtattt tttgtttaat agtgagttta agaattggtt aaatgataat 2760 attgygaata tttgggtgtt ttttttttta atttttttgt tttgttttgt ttttagtttg 2820 tgtgttagtg agtttgattt ttttttgttg aatgttttta aaggatttag tagttaatat 2880 ttttgtttgt tagtttttga tagttatttt tgatagttat ttggtatttt attatatggt 2940 tgtgttaaaa atgatttaat tttttggtta tttgtggatt tgtttattta ygtattaatt 3000 tttagttttt ttatttagta aatattattg gatatgtagt tttgtgtatt gttgtgtttt 3060 tatggaagga tatatttatg tgttgataat ggagtattgg gygtgttgtg tgtaatttgt 3120 tttttattgt tttttttaat tttattttat ttgttgatgt aaggtatgtt ttatgaagtg 3180 gtatgtataa agttttagtt tgggttgtag agttttttgt atatttggyg gttgggaaaa 3240 gagttaggta gatttttaaa ggtgttatat ttgttgtagg agagagttaa atggtttagt 3300 tttggaaagg ttttatttta ggttggggtt atgtaaatga tgtaggtttt ttatgttttt 3360 tggtttgagg gaagtttttt ttgtttttga ggtaatatgt gggttttagt tattagaata 3420 gtttttagta tttttgtttt agatttgtat ttgtagtaaa atagaattat tggatgttta 3480 ggggaaatgg gagggaaagg attattggtt tttttttgtt ttgtagtagt taaagttggt 3540 tttttggggt tgggtgagga tttttttgtt tttttttttt ttttattttt ttggtgggta 3600 gagggtaagt aggtggggat ttgtaatttg gtattttagg aattgtgagg taagtttagt 3660 tttttatgtt tgtttttttt ttttttagtt ttatygtygt aggattttgt ttttttttaa 3720 gagaggaatt agaaggagga tttattagtt tttgaaattt taaaagttat gttttttgtg 3780 agtttttaaa ttttataaat atttgttttt tttattttga aattttttat ttgggtttta 3840 tttagaaatt tggtttttag atttttttat tttagggaat aaaaggatta ggtaggagtt 3900 ttttttttta ttgttttttt ttttttgata aatgtttata gatttatttt ttatagtatt 3960 ttgtattttt tgatggtttt ygtttgttag gaggagaaat ttattttttt gtatgaaatg 4020 agagtatttt ttgtaagaat tgaagtttga agagatggaa tgaatgttta ggagtaatta 4080 tatatagaaa agtaggggag gtggtgttgg ttggttttag ttttagygta tggtatagtg 4140 ttttgttttg ggaatgatta atgattggat atgtaattga ggtttggttg ggaatttatt 4200 aaaaatttga tgttagaatt aaagtttagt ttgaygttgg atagagtatt tgaatttggg 4260 atatttttta ttagtttttt tagtttaaga ggaagaaaag tatagagttg aggatggtga 4320 gygatgtagt aagtgagatg attatttagg tgggtgagtg gttagggttt ggaaaagttt 4380 tagagatttg atttaattty gggatttaaa ataagaggat gtttgagtta gttgttttat 4440 ttgatagaaa attgtgtatt taataaaagt tatttttatg gtttttttta tgaagaaaaa 4500 tgtttgtgtt atgttttaat ataatgatga atttttattt ttaagaaatt tttgatttat 4560 atgaaaattt tgtatttaag atagaaaata tgtttttata attttaaaaa ataaaatatt 4620 tggggaaaat ttattagaaa tattttttgt tgttattgtt attttaaaag atagaagtga 4680 taggttttat tttgagaatt tattgtatgt taagtataga gtgggtagtg tgttttatta 4740 tattgttttt taataataag gaaaatttta tttagtttga ttttaatttt tattttagat 4800 ttgtatatag agtattgatt tgtagttaaa aggaatgttt gagaataatt tgattatttt 4860 gttttattta tttttatttt ttttgttatt ttttatattt agttttattt tttttgttta 4920 ataaaataat ttttttttty gtaaagattt gttttatttt tttttatttt attgtgaatt 4980 attgaaatat atatatatta 5000

Claims (13)

A method for detecting a methylation rate of a target gene as a test marker, in order to provide information necessary for a cancer test, the method comprising the steps of:
In step 1, provide a specimen;
In step 2, the methylation rate of the CpG sequence is detected in at least one target gene of the specimen, wherein the target gene is selected from the group consisting of PTPRR (protein tyrosine phosphatase, receptor type R), ZNF582 (Zinc Finger Protein 582), and PDE8B 8B); &lt; / RTI &gt;
In step 3, the methylation rate of the target gene is detected as the test marker using the methylation rate of the target gene as the test marker. The method according to claim 1,
Wherein said specimen is an isolated specimen selected from the group consisting of cervical smears, ascites, blood, urine, feces, sputum, oral mucous cells, gastric juice, bile, cervical epithelial cells, And detecting the methylation rate of the target gene as a marker. The method according to claim 1,
As a test method for the methylation state of the CpG sequence of the target gene,
Methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS) , A method for detecting the methylation rate of a target gene as a test marker, including microarray, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing or any combination thereof . The method according to claim 1,
The target gene PDE8B (phosphodiesterase 8B) has the nucleotide sequence of SEQ ID NO: 2,
The target gene PTPRR (protein tyrosine phosphatase, receptor type, R) has a nucleotide sequence of SEQ ID NO: 3,
Wherein said target gene ZNF582 (Zinc Finger Protein 582) has a nucleotide sequence of SEQ ID NO: 4 as a test marker. 5. The method of claim 4,
The methylated bisulfite sequencing of the target gene PDE8B (phosphodiesterase 8B) is recognized by the primers of SEQ ID NOs: 15 and 16,
The target gene PTPRR (protein tyrosine phosphatase, receptor type, R) has a nucleotide sequence of SEQ ID NOS: 17 and 18,
Wherein the target gene ZNF582 (Zinc Finger Protein 582) has a nucleotide sequence of SEQ ID NOs: 19 and 20, and is a test marker and detects the methylation rate of the target gene. The method according to claim 1,
The target gene PDE8B (phosphodiesterase 8B) is recognized by the primers of SEQ ID NOS: 7 and 8,
The target gene PTPRR (protein tyrosine phosphatase, receptor type, R) has a nucleotide sequence of SEQ ID NOS: 9 and 10,
Wherein the target gene ZNF582 (Zinc Finger Protein 582) has a nucleotide sequence of SEQ ID NOs: 11 and 12, and is a test marker and detects the methylation rate of the target gene. 7. The method according to any one of claims 1 to 6,
Wherein the cancer is selected from the group consisting of a cervical cancer, a colon cancer and an ovarian cancer. A method for detecting a methylation rate of a target gene as a test marker, in order to provide information necessary for a cancer test, the method comprising the steps of:
In step 1, provide a specimen;
In step 2, the methylation rate of the CpG sequence is detected in at least one target gene of the specimen, wherein the target gene comprises DBC1 (deleted in bladder cancer protein 1);
In step 3, the methylation rate of the target gene is used as the test marker,
Wherein the cancer is selected from the group consisting of a cervical cancer, a colon cancer and an ovarian cancer. 9. The method of claim 8,
Wherein said specimen is an isolated specimen selected from the group consisting of cervical smears, ascites, blood, urine, feces, sputum, oral mucous cells, gastric juice, bile, cervical epithelial cells, And detecting the methylation rate of the target gene as a marker. 9. The method of claim 8,
As a test method for the methylation state of the CpG sequence of the target gene,
Methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS) , A method for detecting the methylation rate of a target gene as a test marker, including microarray, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing or any combination thereof . 9. The method of claim 8,
Wherein the target gene DBC1 (deleted in bladder cancer protein 1) has a nucleotide sequence of SEQ ID NO: 1, and is a test marker and detects the methylation rate of the target gene. 12. The method of claim 11,
Wherein the methylated bisulfite sequencing of the target gene DBC1 (deleted in bladder cancer protein 1) is detected by the primers of SEQ ID NOS: 13 and 14, and detecting the methylation rate of the target gene. 9. The method of claim 8,
Wherein the target gene DBC1 (deleted in bladder cancer protein 1) is a test marker that is recognized by the primers of SEQ ID NOS: 5 and 6, and detects the methylation rate of the target gene.

Images